Universal RT MicroRNA PCR Manual

miRCURY LNA™ Universal RT
microRNA PCR
Instruction manual v6.2
#203301-203351
July 2016
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Table of contents
Product summary 3
I. Reagent kits 4
II. Primer Sets and Panels 6
Storage 10
Additional required materials 10
Recommended accompanying products 11
Product description 12
Control Assays 13
Before starting the experiment 17
Protocols 20
A. Individual assays 20
B. Human and Mouse&Rat microRNA PCR Panels 29
C. Focus microRNA PCR Panels 36
D. Pick-&-Mix microRNA PCR Panels 43
Tips to protocol 52
Troubleshooting guide 60
FAQs 61
Related products 63
Product summary
The miRCURY LNA™ Universal RT microRNA PCR system is a microRNA-specific,
LNA™-based system designed for sensitive and accurate detection of microRNA by
quantitative real-time PCR using SYBR® Green. The method is based on universal reverse
transcription (RT) followed by real-time PCR amplification with LNA™ enhanced primers
(for more details please see page 12). The miRCURY LNA™ Universal RT microRNA PCR
portfolio is comprised of four types of reagent kits; including:
• Universal cDNA synthesis kit II
• RNA Spike-in kit
• ExiLENT SYBR® Green master mix kit
• microRNA primer sets available in pre-defined Human, Mouse&Rat and Focus PCR panels and
customized Pick-&-Mix PCR panels as well as individual primer sets and reference genes. All PCR
panels are Ready-to-Use delivered with one 10 μL PCR reaction per well.
Product overview
Universal cDNA synthesis kit II RNA Spike-in kit

Description page 4 (optional)
+
microRNA and reference Pick-&-Mix PCR panels Predefined PCR panels
gene primer sets (Ready-to-Use) Human, Mouse&Rat, and Focus PCR panels
(Ready-to-Use)
Description page 6, Description page 9, Description page 7,

protocol page 20 protocol page 43 protocol page 29+36
+
ExiLENT SYBR® Green master mix
1, 2,5 or 20ml
Figure 1.
Description on page 5
3
I. Reagent kits
Universal cDNA synthesis kit II, 8-64 rxns (product # 203301)

This kit contains all reagents required for first-strand cDNA synthesis for 8-64 reactions1).
The UniSp6 RNA Spike-in template can be used by itself or in combination with the cel-miR-
39-3p template provided in the RNA Spike-in kit:
Contents Amount supplied

5× Reaction buffer 128 μL, 5x concentrated
Enzyme mix 64 μL, 10x concentrated
Nuclease free water 1 ml
UniSp6, RNA Spike-in template3) 12 fmol, dried down
Table 1.
miRCURY LNA™ Universal RT microRNA PCR, Starter Kit (product # 203351)

This kit contains all reagents required to perform 20 cDNA reactions of 10 μL volume and
100 PCR reactions. Kit includes primer set for the spike control UniSp6 (included), one
candidate endogenous control primer set (miR-103a-3p) and two validated primer sets of
your choice.

5x Reaction buffer2) 128 μL, 5x concentrated
Enzyme mix 24 μL, 10x concentrated
UniSp6, RNA Spike-in template 12 fmol, dried down
UniSp6 RNA Spike-in control primer set v24), lyophilized 200 rxn
hsa-miR-103a-3p primer set (also works for mmu+rno),
200 rxn
lyophilized
2 primer sets free of choice from stocked primers 2x 200 rxn
ExiLENT SYBR® Green master mix, 2x concentrated 500 μL
Nuclease free water 1.25 mL
Table 2.
1) Number of reactions is based on a standard reaction volume of 10 μL to 80 μL. Reaction volume depends on the application
and number of assays to profi le. Please consult Figure 4 for details.
2) Includes universal reverse transcription primer.
3) Used exclusively for the UniSp6 RNA Spike-in control primer set included in the ExiLENT SYBR® Green master mix kit.
4
upplied
Note:
p r im e r se t n ow s in 2 2 0 μL
U niSp6 d - R e-su spend se!
e
lyo philiz free water before u
nu c le a s e
ExiLENT SYBR® Green master mix
These kits contain all reagents required for PCR amplification of microRNAs. In addition,
a positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this
kit for amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA
synthesis kit II.
ExiLENT SYBR® Green master mix, 1 ml (product # 203401). The master mix is provided in
an amount sufficient for 200 reactions of 10 μL:

ExiLENT SYBR® Green master mix, 2x concentrated 2x 0.5 ml, 2x concentrated
Nuclease free water 1x 1.25 ml
Table 3.
ExiLENT SYBR® Green master mix, 2.5 ml (product # 203403). The master mix is provided
in an amount sufficient for 500 reactions of 10 μL:

ExiLENT SYBR® Green master mix, 2x concentrated 2x 1.25 ml, 2x concentrated
Nuclease free water 2x 1.25 ml
Table 4.
ExiLENT SYBR® Green master mix, 20 ml (product # 203421). The master mix is provided in
an amount sufficient for 4000 reactions of 10 μL:

ExiLENT SYBR® Green master mix, 2x concentrated 2x 10 ml, 2x concentrated
Nuclease free water 1x 20 ml
Table 5.
1) Used exclusively for detection of the UniSp6 RNA spike-in provided with the Universal cDNA synthesis kit II.
5
II. Primer Sets and Panels
MicroRNA LNA™ PCR primer set (product # 204000-206xxx and 2100000-21xxxxx)

LNA™ PCR primer sets are designed for optimal performance with the Universal cDNA
Synthesis Kit II and the ExiLENT SYBR® Green master mix, kit II. The performance of LNA™
primer sets will be affected if used in combination with less than optimal reagents. The
primer sets are supplied in sufficient amounts for 200 reactions of 10 μL.
Contents
LNA™ PCR Primer set (dried down)
Table 6.
Reference gene primer set (product # varies)

The Reference gene primer sets are designed for use with the microRNA primers sets above
as reference genes for normalization. The primer set is supplied in sufficient amount for 200
reactions of 10 μL.
Contents
LNA™ PCR Primer set (dried down)
Table 7.
6
Pre-defined microRNA PCR Panels (product # varies)

The Human, Mouse&Rat and Focus microRNA PCR panels Ready-to-Use consist of
either 96-well or 384-well PCR plates containing dried down LNA™ primer sets for one
10 μL real-time PCR reaction per well. The LNA™ primer sets are designed for optimal
performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR® Green master
mix, kit. The performance of the LNA™ primer sets will be affected if they are used in
combination with less than optimal reagents.
Contents Human miRNome panel I and II, V4

384-well PCR plates supplied with LNA™ primer sets dried down, one 10 μL reaction per well:
Panel I Panel II
372 LNA™ primer sets for the 380 LNA™ primer sets for the
amplification of human microRNAs1) amplifi cation of human microRNAs1)
3 inter-plate calibrators 3 inter-plate calibrators
3 primer sets for reference genes2) 1 blank well
5 RNA Spike-in control primer sets3)
1 blank well
Table 8.
Contents Mouse&Rat miRNome panel I and II, V4

384-well PCR plates supplied with LNA™ primer sets dried down, one 10 μL reaction per well:
Panel I Panel II
372 LNA™ primer sets for the 380 LNA™ primer sets for the
amplification of mouse and rat microRNAs1) amplifi cation of mouse and rat microRNAs1)
3 inter-plate calibrators 3 inter-plate calibrators
2)
3 primer sets for reference genes 1 blank well
5 RNA Spike-in control primer sets3)
1 blank well
Table 9.
7
Contents Serum/Plasma Focus microRNA PCR panel, V4

PCR plates compatible with various real-time PCR instruments are available and supplied
with LNA™ primer sets dried down, one 10 μL reaction per well:
96-well (2 plates) 384-well plate (2 panels per plate)

179 LNA™ primer sets for the amplification 2x179 LNA™ primer sets for the amplification
of human microRNAs1+2) of human microRNAs1+2)
2x3 Inter-plate calibrators 2x6 Inter-plate calibrators
5 RNA Spike-in control primer sets3) 2x5 RNA Spike-in control primer sets3)
2 blank wells - 1 in each plate 2x2 blank wells
Table 10.
Contents Cancer Focus microRNA PCR panel, V4

PCR plates compatible with various real-time PCR instruments are available and supplied
with LNA™ primer sets dried down, one 10 μL reaction per well:
96-well (1 plates) 384-well plate (4 panels per plate)

84 LNA™ primer sets for the amplification 4x84 LNA™ primer sets for the amplification
of human microRNAs1) of human microRNAs1)
3 Primer sets for potential 4x3 Primer sets for potential
reference genes2) reference genes2)
3 Inter-plate calibrators 4x3 Inter-plate calibrators
5 RNA Spike-in control primer sets3) 4x5 RNA Spike-in control primer sets3)
1 blank well 1 blank well
Table 11.
1) Please go to www.exiqon.com/mirna-pcr to download plate layout files.

2) Human Panels and Cancer Focus Panel: Three snRNAs (U6snRNA, SNORD38B, SNORD49A). Mouse&Rat Panels: Three snRNAs
(U6snRNA, RNU5G, RNU1A1). Serum/plasma Focus Panel: miR-103a-3p, miR-191-5p, miR-423-5p, miR-16-5p, miR-425-5p,
miR-93-5p, miR-451a are regarded reference gene candidates.
3) The RNA Spike-in control primer sets targets the UniSp6 RNA spike-in supplied in the Universal cDNA synthesis kit II and the 4 RNA
spike-ins contained in the RNA Spike-in kit (UniSp2, UniSp4, UniSp5, and cel-miR-39-3p).
8
Pick-&-Mix microRNA PCR Panel, (product # 203893-203897 and 203818-203819)

The Pick-&-Mix microRNA PCR Panels consist of either 96-well PCR plates or 384-well
PCR plates containing custom selections of dried down microRNA LNA™ PCR primer sets
for one 10 μL real-time PCR reaction per well, ready-to-use. PCR plates compatible with
various real-time PCR instruments are available. The LNA™ primer sets are designed for
optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR® Green
master mix kit. The performance of the LNA™ primer sets will be affected if they are used
in combination with less than optimal reagents.
Contents
PCR plates supplied with customer defined LNA™ primer sets, reference gene primer sets,
and RNA Spike-in control primer sets, dried down, one 10 μL reaction per well1):
96-well PCR plates1) 384-well PCR plates1)

10 primer sets in 8 replicates 22 primer sets in 16 replicates
22 primer sets in 4 replicates 46 primer sets in 8 replicates
92 primer sets in 1 replicate 94 primer sets in 4 replicates
96-well flexible layout 380 primer sets in 1 replicate
384-well flexible layout
Table 12.
1) Please go to www.exiqon.com/pick-and-mix to configure a Pick-&-Mix microRNA PCR Panel.
9
Storage
All microRNA PCR Panels, LNA™ primer sets and Reference gene primer sets
The PCR panels and primer sets are shipped dried down at room temperature. The primers
can be stored between +4°C and -20°C. Under these conditions, all components are stable
for at least 12 months. Aer resuspension, it is recommended to store LNA™ primer sets
and Reference gene primer sets in aliquots at -20°C to avoid repeated freeze-thaw cycles.
Universal cDNA synthesis kit II and ExiLENT SYBR® Green master mix
These kits are shipped on dry ice in polystyrene containers and should be stored at -15°C
to -25°C. Do not store in a frost-free freezer. Under these conditions, all components are
stable until the expiry date on the package or vial. It is recommended that the RNA spike-in
be stored in aliquots at -20°C aer re-suspension to avoid repeated freeze-thaw cycles.
Additional required materials
Reagents not supplied

• ROX or other passive reference dye (required on some PCR cyclers) see Tip 8 on page 54
for more. See tip 8
Materials and Equipment not supplied

• Nuclease-free PCR tubes or plates for use with individual assays
• Nuclease-free, aerosol barrier pipette tips
• Nuclease-free, low nucleic acid binding microcentrifuge tubes
(e.g. Eppendorf DNA LoBind tubes product and original NUNC vials)
• Sealing foils for PCR plates
• Micro-centrifuge and plate centrifuge
• Heating block, thermal cycler or other incubators
• Real-time PCR instrument
Optional but recommended product

• miRCURY LNA™ Universal RT microRNA PCR, RNA Spike-in kit
(product# 203203)
10
Recommended accompanying products
Exiqon recommends the Exiqon GenEx qPCR soware for comprehensive and convenient
data analysis. GenEx includes a wizard for import of Exiqon miRCURY™ Universal RT
microRNA PCR data and offers advanced methods to analyze real-time qPCR data in a few
simple steps. The soware includes tools for selection and validation of reference genes,
data pre-processing and comprehensive statistical analyses. For more information and to
download a free trial, please go to www.exiqon.com/qpcr-soware. The following Exiqon
GenEx products are available:
Exiqon GenEx6 Industrial - Exiqon version of GenEx,

qPCR analysis soware, industrial license
Exiqon GenEx6 Academic - Exiqon version of GenEx,

qPCR analysis soware, academic license
Exiqon recommends the miRCURY™ RNA Isolation kits for purification of total RNA
or small RNA fraction. RNA purified using the miRCURY™ RNA Isolation kits is fully
compatible with the miRCURY LNA™ Universal RT microRNA PCR System. The following
kits are available:
miRCURY™ RNA Isolation Kit – Cell & Plant

Provides a rapid method for purification of total RNA from cultured animal cells, small
tissue samples, blood, bacteria, yeast, fungi and plants.
miRCURY™ RNA Isolation Kit – Tissue

Specifically designed for purification of total RNA from tissue samples.
miRCURY™ RNA Isolation Kit – Biofluids

Kit for purification of low abundant small RNAs from samples such as serum, plasma, urine
and CSF. See tip 2
11
Product description
A unique system for microRNA profiling
miRCURY LNA™ Universal RT microRNA PCR offers the best available combination of
performance and ease-of-use on the microRNA real-time PCR market because it unites two
important features (Figure 2):
1. Universal RT – One first-strand cDNA synthesis reaction provides template for all
microRNA real-time PCR assays. This saves precious sample, reduces technical variation,
requires use of less reagents, and saves time in the laboratory.
2. LNA™ PCR amplification – Both PCR amplification primers (forward and reverse) are
microRNA specific and optimized with LNA™. The result is: 1) exceptional sensitivity as well
as extremely low background enabling accurate quantification of very low microRNA levels
and 2) highly specific assays that allow discrimination between closely related microRNA
sequences.
miRCURY LNA™ Universal RT microRNA PCR offers solutions both for high-throughput
microRNA expression profiling and for quantification of individual microRNAs.
Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System.
Step 1: First-strand synthesis (RT)

Mature microRNA
A) AAAAAAAAAAAAAAAAAAAA
B) AAAAAAAAAAAAAAAAAAAA
TTTTTTTTTTTTTTT
5’ universal tag
3’ degenerate anchor
Step 2: Real-time PCR amplification

miR-specific forward primer
A) LNA LNA LNA
TTTTTTTTTTTTTTT
LNA LNA LNA
miR-specific reverse primer

B)
Figure 2. A poly-A tail is added to the mature microRNA template (step 1A). cDNA is
synthesized using a poly-T primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B).
The cDNA template is then amplified using microRNA-specific and LNA™-enhanced forward
and reverse primers (step 2A). SYBR® Green is used for detection (step 2B).
12
Control Assays
There are 3 different types of control assays available in the miRCURY LNA™ Universal RT
microRNA PCR system:
• Reference assays and reference candidates
• Inter-plate calibrators
• RNA spike-in assays
All of these control assays are available in the microRNA PCR panels. One RNA spike-in
template is provided with the Universal cDNA Synthesis Kit II, while the assay that will detect this
RNA spike-in is available in the ExiLENT SYBR® Green master mix. Additionally 4 RNA spike-in
templates are available as a spike-in kit. The assays for detection of these 4 templates as well as
the reference assays are available as individual primer sets.
Reference assays and reference candidates

These assays detect small non-coding RNAs - either small nuclear RNA, small nucleolar RNA or
microRNA – which frequently are found to be stably expressed across different cells or tissues.
Reference assays may therefore be candidate assays for normalization in a profiling study with
several samples. Though this is a good and recommended approach, great caution should be
taken in the selection of reference genes. The danger of using endogenous reference genes lies in
the assumption that a specific gene is expressed at the exact same level in all sample types. This
is rarely true. The selection of reference genes should therefore be made with care, and should
be specific to the sample set you are working with. The actual selection of reference genes to be
used for normalization should always be based on a determination of the most stably expressed
gene(s) which may be done using either GeNorm or NormFinder – both tools that are integrated
within Exiqon’s GenEx data analysis soware. When applicable, we recommend using microRNA
rather than small nuclear RNA or small nucleolar RNA for normalization. Firstly, small nuclear and
nucleolar RNAs are longer RNA species than microRNA and may purify differently from microRNA.
Moreover, small nuclear and nucleolar RNAs have entirely different functions as well as sub-
cellular locations, and finally certain samples like blood plasma does not contain the small nuclear
and nucleolar RNAs. Global mean normalization is a preferred alternative to using reference genes
for normalization when working with panels and samples where many microRNAs are screened
per sample and where many microRNA are called (detected) in all samples. Exiqon’s GenEx will
easily perform global mean normalization. To read more on normalization. See tip 11
13
Inter-plate calibrators
Three wells within the pre-defined Human, Mouse&Rat, and Focus PCR Panels contain the
inter-plate calibrator assay (annotated as UniSp3 IPC in the plate layout files). Depending on
the plate layout, the Pick-&-Mix Panels contain at least three inter-plate calibrators. Each
of these wells, contain a pre-aliquoted primer pair and a DNA template and therefore the
variation is very minimal from well-to-well and from plate-to-plate of these assays. The
inter-plate calibrators are used for calibration between PCR plate runs which is very useful
on some instruments that apply the cycle threshold method for Cq determination such as
the ABI7900 PCR cycler. Since the inter-plate calibrators are independent of cDNA quality
in order to give a signal (but may be affected by PCR inhibitors in the sample) they may be
used to quality control each plate run.
Inter-plate calibration (IPC) can easily be performed in the data analysis soware Exiqon’s
GenEx. Alternatively, IPC may be performed manually by using the IPC assay replicates as
follows. For each plate, verify that the replicates have Cq standard deviation within 0.5. If
this is not the case, eliminate the outlier if this can be identified. Calculate the average of
the replicates for each plate, the overall average (average of IPC values from all plates). The
calibration factor is calculated as the difference between plate average and overall average
for each plate (calibration factor = IPCplate-IPCoverall). An example is shown in Table 13.
Finally, calibrate each plate by subtracting the calibration factor from all Cq values in the plate.
Plate 1 Plate 2 Plate 3

let-7c 21.12 20.93 21.34
IPC plate average 19.72 19.70 20.00
IPC overall average 19.81 19.81 19.81
Calibration factor -0.09 -0.11 0.19
let-7c calibrated 21.21 21.04 21.15
Table 13. Example of inter-plate calibration (IPC)
14
RNA spike-ins (synthetic control templates)

The primary purpose of the RNA spike-ins and the matching primer pairs for detection of
these is to provide controls for the quality of the RNA isolation, the cDNA synthesis reaction
and the PCR. RNA isolations may vary in yield, purity and integrity. Some sample types may
contain compounds that inhibit the cDNA synthesis or the PCR even though the RNA has
been purified using the best standard procedures. This may result in different efficiencies
of the reverse transcription or PCR between compared samples. One way to control for
differences in efficiencies at each experimental level (isolation, cDNA synthesis, and PCR) is
by adding known RNA spike-ins to the sample prior to isolation and cDNA synthesis. Use
of the RNA spike-ins may also reveal if nucleases are present. Aer conducting the PCR but
before initiating the data analysis, wells detecting RNA spike-ins are compared and outlier
samples may be identified and considered for exclusion in the further data analysis.
We have designed a flight of RNA spike-ins for this purpose. The UniSp6 RNA Spike-in
template is provided with the Universal cDNA synthesis kit II. Additionally four RNA spike-in
templates can be obtained with the separate RNA Spike-in kit. Here, a vial of three RNA
spike-in templates, UniSp2, UniSp4 and UniSp5, mixed at different concentrations can be
used during RNA isolation. The cel-miR-39-3p RNA template provided in a separate vial
in the RNA Spike-in kit can be mixed with the UniSp6 template from the Universal cDNA
synthesis kit II to obtain two different template concentrations. This combination can be
added during the cDNA synthesis. Five wells in pre-defined PCR panel plates contain the
matching primer sets. The selection of RNA Spike-in control primer set in the Pick-&-Mix
PCR Panel can be customized to the specific need. A UniSp6 control primer set is also
provided with the ExiLENT SYBR® Green master mix kit, which is to be used with our non-
plate based PCR primer set products.The RNA spike-ins are shipped dried down and must
be re-suspended before use.
If the RNA Spike-in kit with multiple RNA spike-ins is used, follow the protocol
accompanying that product.
If UniSp6 is to be used alone:

1. Re-suspend the UniSp6 RNA spike-in by adding 80 μL nuclease free water to the tube.
2. Mix by vortexing and spin down. Leave for 20-30 min. on ice to fully dissolve RNA spike-
in. Mix by vortexing and spin down. Store in aliquots at -20°C
3. Prior to the RT reaction, add 1 μL synthetic spike-in (108 copies/μL) per 20 ng sample RNA.
15
An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This is

only relevant when using individual LNA™ primer sets and Reference gene primer sets in a
multi-plate set-up. The microRNA PCR panels already contain an inter-plate calibrator. Add
1 μL synthetic spike-in (108 copies/μL) to 20 ng of a complex RNA sample
(e.g. total RNA from MS2, yeast, or a cell line; not provided with the kit). Proceed with
first strand synthesis and subsequently real-time PCR as described in the protocols of the
current instruction manual. At least one spike-in amplification reaction per PCR plate is
used for inter-plate calibration. See tip 9
Experimental design
Before starting the experiment, it is essential to consider the experimental setup and
consider the number of replicates needed for obtaining significant results – replicates being
technical as well as biological. The number of biological replicates required varies from
experiment to experiment depending on the variation within and between the groups. We
recommend that a No Template Control (NTC) is included in the study every time a new
experiment is set up, to set the background level. The most optimal NTC is a mock up
sample preparation including only carrier RNA as sample. The NTC should be run on all
assays included in the study. We define the background of an assay as 5 Cq values below
the NTC level. Furthermore we recommend including spike-ins found in the Spike-in kit to
provide full quality control over all steps in the profiling (see figure 3). Finally it is necessary
to include a number of candidate reference miRNAs, which are expected to be constitutively
expressed across the different experimental conditions, for data normalization.
Experimental design
Comes with Assays available as

Included in all individual assays or in
Sold as separate Spike-in kit (203203) Universal cDNA
panel products ready-to-use panels
synthesis kit II
UniSp2 UniSp4 UniSp5 cel-miR-39-3p UniSp6 UniSp3 miR-A miR-X

Sample n
Sample n+1 Sample preparation evaluation cDNA synthesis evaluation Inter plate Calibration Sample profiling
NTC
Figure 3.
16
Before starting the Experiment
Important note
RNA work requires specific handling and precautions to prevent RNase

contamination of the reagents and degradation of the RNA sample. Find information
on how to handle RNA in the tips section starting on page 52. The tips section
also provides simple guidelines for good laboratory practice to ensure optimal
performance of PCR experiments.
Before setting up a real-time PCR experiment, there are a number of practical experimental
design parameters that should be considered:
RNA input - The miRCURY LNA™ Universal RT microRNA PCR protocol is optimized
for use of 20 ng total RNA per 20μL cDNA synthesis reaction. The exact amount of total
RNA needed depends on whether the downstream application is individual assays or
panels. Furthermore, the amount of total RNA to be used may also vary depending on the
microRNA expression levels in the cells or tissue to be analyzed. For highly expressed
microRNAs it is possible to use down to 10 pg total RNA as starting material. For weakly
expressed microRNAs it may be possible to use up to 200 ng of total RNA; however, in
samples with high amounts of PCR inhibitors (e.g. FFPE tissue samples), this may not
be feasible. Finally, inhibitors may be present in RNA preparations from certain samples
e.g. serum and plasma. Prior to conducting a larger microRNA profiling study, it is
recommended to optimize the amount of input RNA to the RT reaction in order to avoid
conducting a larger study where inhibition occurs sporadically throughout the data set.
Information on how to extract and handle RNA can be found in the tips section. In short,
total RNA should be prepared using a method that preserves small RNA species. DNase
treatment may be necessary. When using commercially available kits, please ensure that
the total RNA preparation is guaranteed to contain microRNAs.
17
Note: Blood serum and plasma are particular sample types that require special RNA
purification procedures and the amount of RNA present in the samples can usually not be
accurately determined. Due to the low levels of microRNAs and potentially high levels of
inhibitors in samples derived from serum and plasma, specific recommendations for how
to set up experiments using these types of sample can be found in the miRCURY LNA™
Universal RT microRNA PCR, Instruction Manual “Biofluid samples”.
Normalization – when running individual assays or when configuring a Pick-&-Mix

microRNA PCR panel it is important to consider how the data will be normalized. For tips
and recommendations on choosing the correct reference genes and how to test and validate
reference genes, please see section on reference assays (page13) and the Tip 11 (page 58).
See tip 11
Excess volumes required for pipetting – Liquid handling with pipettes or pipetting robots
require excess volumes of reagents due to loss during pipetting. The loss depends on the
available pipetting system but losses in the range from 10% -25% are not uncommon.
All protocols in the current instruction manual reflect the required reaction volumes and
pipetting volumes should be adjusted according to accommodate the pipetting loss of the
available pipetting system.
ROX – ROX is a passive reference dye used by some PCR cycles to obtain a robust read
over the entire array of wells in a 96- or 384-well PCR plate. The requirement for ROX
is instrument dependent and we recommend to follow the instrument manufactures
guidelines on this. See tip 8
ABI instruments – the default settings on ABI real-time PCR cyclers are not suitable for
running miRCURY LNA™ Universal RT microRNA PCR. Settings need to be changed from
automatic to manual background and threshold settings to obtain valid PCR data (see
also Tip 10). Furthermore, if the dataset is to be analyzed using the GenEx soware, it is
important that the experiment is set up as an AQ experiment, not RQ. To ensure correct
settings, download the instrument settings file at www.exiqon.com/sds. See tip 10
18
RNA spike-ins – consider how the RNA spike-ins should be applied in the planned study.
Please consult the section on RNA spike-ins on page 15.
Protocols for the first-strand cDNA synthesis and real-time amplication follows on the
next pages:
A. Individual assays, please go to page 20
B. Human and Mouse&Rat microRNA PCR Panels, please go to page 29
C. Focus microRNA PCR Panels, please go to page 36
D. Pick-&-Mix microRNA PCR Panels, please go to page 43
Figure 4 gives an overview and helps identifying which protocol to follow as well as the
recommended cDNA reaction volume needed for a given sample and assay type.
Protocol overview
Serum plasma or other biofluids
www.exiqon.com/pcr-manual-biofluids
Use biofluids manual from
Sample type Cells or tissue
Pick-&-Mix/Focus Individual
Panels or miRNome miRNome
assays
primer sets: panel I panel I+II (<96)
1-96 97-192
Universal cDNA
32 16 64 32 64
reactions per kit
Universal cDNA
reaction volume 20 µL 40 µL 10 µL 20 µL 10 µL
Dilution of cDNA
in ExiLENT 100x 80x
Master Mix
Use standard printed manual or download from

www.exiqon.com/pcr-manual
Figure 4.
19
Protocol A - Individual assays

This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR,
using the individual assays for human, mouse and rat (product numbers 204000-206xxx).
If working with serum plasma samples or other biofluids, please refer to the specific
miRCURY LNA™ Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Before using the LNA™ PCR primer set , the UniSp6 RNA spike-in control primer set or
the Reference gene primer set for the first time, the primers must be re-suspended:
• Re-suspend the primer set by adding 220 μL nuclease free water to the tube. Mix by
vortexing and spin down. Leave on ice for 20-30 minutes.
Additional required materials:

• 96- or 384-well plate real-time PCR cycler
• Thermocycler for first-strand cDNA synthesis
• 96/384-well plates or tube strips compatible with available real-time PCR cycler
• Micro centrifuge
• Swing bucket centrifuge for 96-/384-well plates
Individual assays
Protocol A.
20
Checklist:
• Have you considered excess volumes required for using liquid handling robotics? – see page 18
• Did you consider how to use the RNA spike-ins? – please see page 15
• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference
dye. Please follow instrument manufactures recommendations
• ABI instruments: The use of manual background and threshold settings is necessary
for obtaining correct PCR data. Make sure to have the optimal settings by downloading
the instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be
analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ
• Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Individual assays
Protocol A.
21
Workflow for individual primer sets (per sample)
Phase I: Prepare RNA sample

See page 52 for recommendations
Phase II: cDNA synthesis

See protocol page 23.
Phase III: real-time PCR amplification

- Prepare adequate amount of pre-mixed primer
+ PCR Master mix and distribute into wells
- Add cDNA to all primer sets to be analyzed
Individual assays
LNATM primer sets

Protocol A.
4
Phase IV: Data analysis
Relative expression (log2)
2 See data analysis guide online

1
0
- Export data for further analysis
-1
Normal Tumor Total Tumor Tumor
- Data pre-processing, normalization
miR-21
let-7a
stroma
and statistical analysis
22
Protocol
The miRCURY LNA™ Universal RT microRNA PCR protocol is a two-part protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-11)
Important: Keep reagents and reactions on ice (or at 4˚C) at all times.
First strand synthesis
Step 1 Adjust each of the template RNA samples to a concentration of

Dilute template RNA 5 ng/μL using nuclease free water.
Step 2 Gently thaw the 5x Reaction buffer and nuclease-free water, and
Prepare reagents immediately place on ice. Mix by vortexing. Re-suspend the RNA
spike-ins according to the appropriate RNA Spike-ins protocol (see
page 15), leave on ice for 15-20 minutes. Immediately before use,
remove the Enzyme mix from the freezer, mix by flicking the tubes and
place on ice. Spin down all reagents.
Individual assays
Protocol A.
23
Step 3 If performing first-strand cDNA synthesis on multiple RNA samples, it

Combine reagents is recommended to prepare an RT working solution of the 5x Reaction
according to Table 14 buffer, water, Enzyme mix and RNA spike ins (in the proportion
indicated in the first four lines of Table 14).
Note: remember to The following procedure is recommended:
calculate necessary 1. Prepare the required amount of RT working solution and place it on ice.
excess volume for 2. Dispense RT working solution into nuclease free tubes.
pipetting and robotic 3. Dispense template RNA in each tube.
dead volume
Reagent Volume (μL), RT reaction
5x Reaction buffer 2
Nuclease-free water 4.5
Enzyme mix 1
Synthetic RNA spike ins, optional
0.5
replace with H2O if omitted
Template total RNA (5 ng/μL) 2
Total volume 10
Table 14. Reverse transcription reaction setup
Step 4 Mix the reaction by very gentle vortexing or pipetting to ensure that all
Mix and spin reagents are thoroughly mixed. After mixing, spin down.
reagents
Step 5 • Incubate for 60 min at 42˚C.

Individual assays
Protocol A.
Incubate and heat • Heat-inactivate the reverse transcriptase

inactivate1) for 5 min at 95˚C.
• Immediately cool to 4°C.
• Store at 4°C or freeze.
1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks (optional store at 4˚C for up
to 4 days). It is recommended that synthesized cDNA is stored in “low-nucleic acid binding” tubes or plates.
24
qPCR protocol
Step 6 Place cDNA (from Step 5), nuclease free water and PCR Master mix on
Prepare reagents for ice and thaw for 15-20 min. Protect the PCR Master mix vials from light.
real-time PCR Immediately before use, mix the PCR Master mix by pipetting up and
down. The rest of the reagents are mixed by vortexing and spun down.
Step 7 Immediately before use, dilute only the amount of cDNA template
Dilute cDNA template needed for the planned real-time PCR reactions 80x in nuclease free
80x in nuclease water (e.g. add 395 μL nuclease free water to each 5 μL of reaction). It
free water2) is important that “low-nucleic acid binding” tubes or plates are used. It
is not recommended to store the 1:80 dilution of cDNA.
Recommendation: Include a passive reference dye in the cDNA

dilution if advised by instrument manufacturer. Please note that the
PCR Master mix does not include ROX. The amount of ROX required is
instrument dependent and it is important to refer to the manufacturer’s
recommendations when deciding how much ROX to use.
See tip 8
2)
Adjust volumes to accommodate your in-house liquid handling system volume loss when pipetting
Individual assays
Protocol A.
25
Step 8 When multiple real-time PCR reactions are performed with the same
Combine PCR Master microRNA primer set, it is recommended to prepare a primer master
mix, PCR primer set mix working-solution of the PCR primers and the PCR Master mix (in
and cDNA according the proportion indicated in Table 15).
to Table 15 The following procedure is recommended:
1. Prepare the required amount of primer:master mix working-
Mix thoroughly solution (see Table 3) and place it on ice. It is recommended to
include excess of all reagents in the master mix to compensate for
Note: remember to pipetting excess material.
calculate necessary 2. Place the relevant volume of primer:master mix working-solution
excess volume for in PCR tubes/wells (see Table 15) and spin tubes/plate briefly in a
pipetting and robotic centrifuge (1500g for 1 minute), to remove air bubbles.
dead volume 3. Add cDNA template to each tube/well.
Volume (μL), 96/384-well

Reagent
plate, tubes or strips
PCR Master mix 5
4)
PCR primer set 1
Diluted cDNA template 4
Total volume 10
Table 15. Real-time PCR reaction, pr. 10 μL reaction3)
Step 9 Mix the reaction by gentle pipetting to ensure that all reagents are
Mix and spin mixed thoroughly. After mixing cap tubes or strips or seal the plate
reagents with optical sealing as recommended by the manufacturer. Spin down
in a centrifuge (1500g for 1 minute). The experiment can be paused at
this point. Store the reactions protected from light at 4°C for up to 24
Individual assays
hours.
Protocol A.
3) If using a 96-well cycler with a minimum recommended volume of 20 μL (as some ABI instruments), then use 10 μL reaction volume
and set the instrument settings at 20 μL.
4) The PCR primer set must be dissolved prior to real-time PCR set-up, see page 20.
26
ABI instrument user?

Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file
Step 10 Perform real-time PCR amplification followed by melting curve

Real-time PCR analysis according to Table 16.
amplification
Settings, LC480 Settings, other
Process step
instrument5) instruments3)
Polymerase Activation/ 95˚C, 10 min 95˚C, 10 min
Denaturation
Amplification 45 amplification 40 amplification
cycles at cycles at
95˚C, 10 s 95˚C, 10 s
60˚C, 1 min, 60˚C, 1 min,
ramp-rate ramp-rate
1.6˚C/s6) 1.6˚C/s6)
Optical read Optical read
Melting curve analysis7) Yes Yes
Table 16. Real-time PCR cycle conditions
Step 11 Perform initial data analysis using the software supplied with the real-
Analyze data time PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that it
is not recommended to use auto Ct settings. Furthermore, if the data is
to be analyzed using Exiqon GenEx, the experiment must be set up as
Individual assays
an AQ experiment, not RQ. For a guide on how to set manual baseline

Protocol A.
and threshold, refer to Tip 10, page 56 in the tips section. If you are
using a Roche LC480 instrument, we recommend analysis using the
2nd derivative method.
For tips on normalization, please see Tip 11, page 58. We recommend
performing normalization and further data analysis with the Exiqon
GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis).
Please refer to our data analysis guide
for recommendations.
See tip 11
27
5) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up
to 40.
6) The ramp-rate of cooling from 95˚C to 60˚C should be set to 1.6°C/s. This is equivalent to 100% under standard cycling conditions on
the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised.
7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction. Melting
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier.
Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
Individual assays
Protocol A.
28
Protocol B - Human and Mouse&Rat

microRNA PCR Panels
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR
using the following products:
• Human miRNome PCR Panel (product numbers 203611 to 203618)
• Mouse&Rat miRNome PCR Panel (product numbers 203709 to 203716)

• 384-well plate real-time PCR cycler
• Tube for mixing water and master mix (10 ml)
• Swing bucket centrifuge for 96/384-well plates
• Recommended: Liquid handling robot for pipetting
Checklist:
• Have you considered excess volumes required for using liquid handling robotics? –
see page 18
• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference
dye. Please follow instrument manufactures recommendations Mouse&Rat microRNA PCR Panels
• ABI instruments: The use of manual background and threshold settings is necessary for
obtaining correct PCR data. Make sure to have the optimal settings by downloading the
Protocol B. Human and
instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed

using GenEx, the experiment must be set up as an AQ experiment, not RQ.
29
Workflow for Human and Mouse&Rat microRNA PCR Panels (per sample)



- Mix cDNAs with PCR Master mix
- Add cDNA:PCR Master mix to
PCR plates
Mouse&Rat microRNA PCR Panels
4

1
0
-1
miR-21
let-7a
stroma
30
Protocol
First strand synthesis:
Step 1 Adjust each of the template RNA samples to a concentration of 5 ng/μl

Dilute template RNA using nuclease free water.
spike-in(s) according to the appropriate RNA Spike-in protocol (see

31
Step 3 If performing first-strand cDNA synthesis on multiple RNA samples, it

Combine reagents is recommended to prepare an RT working solution of the 5x Reaction
according to Table 17 buffer, water, Enzyme mix and RNA spike ins (in the proportions
indicated in the first four lines of Table 17).
dead volume.
Panel I Panel I+II
Reagent Volume Volume
(μL) (μL)
5x Reaction buffer 4 8
Nuclease-free water 9 18
Enzyme mix 2 4
Synthetic RNA spike ins, optional 1 2
Template total RNA (5ng/μL) 4 8
Total volume 20 40
reagents
Step 5 • Incubate for 60 min at 42°C.

Incubate and heat • Heat-inactivate the reverse transcriptase

inactivate1) for 5 min at 95°C.
32
qPCR protocol:
Step 6 Place cDNA (from Step 5), nuclease free water and PCR Master mix
Prepare reagents for on ice and thaw for 15-20 min. Protect the PCR Master mix vials from
real-time PCR light. Immediately before use, mix the PCR Master mix by pipetting up
and down. The rest of the reagents are mixed by vortexing and spun
down.
Step 7 The following procedure is recommended to avoid low concentrations

Combine PCR Master of cDNA from adhering to tube surface:
mix, water and cDNA 1. Before removing the plate seal, briefly spin
and add to PCR plates2) down the plate(s) in a plate centrifuge.
2. Combine 2x PCR Master mix and water. Panel I: 2000 μL 2x master
Mix thoroughly mix and 1980 μL water, Panel I+II: 4000 μL 2x master mix and 3960
μL water.
3. Mix gently and spin down.
4. Add 20 μL cDNA (panel I) or 40 μL cDNA (panel I+II) and mix.
5. Add 10 μL PCR Master mix: cDNA
mix to each well3).
6. Seal the plate with optical sealing as recommended by the
instrument manufacturer.
7. Spin plate briefly in a plate centrifuge (1500g for
1 minute), to to collect the sample.
The experiment can be paused at this point. Store the reactions

protected from light at 4°C for up to 24 hours.


See tip 8
33

Step 8 Perform real-time PCR amplification followed by melting curve analysis

Real-time PCR according to Table 18.
amplification
Settings, LC480 Settings, other
Process step
instrument 4) instruments
Denaturation
cycles at cycles at
95˚C, 10 s 95˚C, 10 s
60˚C, 1 min, 60˚C, 1 min,
ramp-rate ramp-rate
1.6˚C/s5) 1.6˚C/s5)
Step 9 Perform initial data analysis using the software supplied with the
Analyze data real-time PCR instrument to obtain raw Cq values (Cp or Ct, depending
on PCR instrument). If you are using an ABI instrument, please note
that it is not recommended to use auto Ct settings. For a guide on how

to set manual baseline and threshold, refer to Tip 10, page 56 in the
tips section. Furthermore, if the data is to be analyzed using Exiqon

GenEx, the experiment must be set up as an AQ experiment, not
RQ. Alternatively, use ABI settings files available from www.exiqon.
com/sds. If you are using a Roche LC480 instrument, we recommend
analysis using the 2nd derivative method.
Please refer to our data analysis guide for recommendations.
See tip 11
34
2) Adjust volumes to accommodate your in-house liquid handling system and the inaccuracy these have when pipetting.
3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.
4) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up
to 40.
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier. Note:
The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.

35
Protocol C - Focus microRNA

PCR Panels
using the Cancer Focus microRNA PCR panels or any of the Toxicology Focus microRNA
PCR Panels, 96-well plates and 384-well plates.

• Swing bucket centrifuge for 96/384-well plates
Checklist:
• ROX: The ExiLENT SYBR® Green master mix does not include the ROX passive reference dye.
Please follow instrument manufactures recommendations
• ABI instruments: The use of manual background and threshold settings is necessary for
obtaining correct PCR data. Make sure to have the optimal settings by downloading the
Focus microRNA PCR Panels
instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed

using GenEx, the experiment must be set up as an AQ experiment, not RQ
Protocol C.
36
Workflow for Focus microRNA PCR Panels (per sample)



- Add cDNA:PCR Master mix to
PCR plates

Protocol C.
4

1
0
-1
miR-21
let-7a
stroma
37
Protocol
The miRCURY LNA™ Universal RT microRNA PCR protocol is a two-part
protocol consisting of:
Step 1 Adjust each of the template RNA samples to a concentration of 5 ng/

Dilute template RNA μL using nuclease free water.
Protocol C.
38
Step 3 When performing first-strand cDNA synthesis on multiple RNA

Combine reagents samples, it is recommended to prepare an RT working solution of
according to Table 19 the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the
proportions indicated in the first four lines of Table 19).
dead volume
Reagent Per panel Volume (μL)
5x Reaction buffer 2
Nuclease-free water 4.5
Enzyme mix 1
Synthetic RNA spike ins, optional 0.5
Template total RNA (5ng/μL) 2
Total volume 10
reagents

Incubate and • Heat-inactivate the reverse transcriptase
heat inactivate1) for 5 min at 95˚C.
Protocol C.

39
qPCR protocol:
Prepare reagents on ice and thaw for 15-20 min. Protect the PCR Master mix vials from
for real-time PCR light. Immediately before use, mix the PCR Master mix by pipetting up
down.
Step 7 The following procedure is recommended to avoid low concentrations

Combine PCR Master of cDNA from adhering to tube surface:
mix, water and cDNA 1. Before removing the plate seal, briefly spin down the plate(s) in a
and add to PCR plates2) plate centrifuge.
2. Combine 2x PCR Master mix and water: 1000 μL 2x master mix and
Mix thoroughly 990 μL water
(Focus panel consisting of 2x 96 assays)
3. Mix gently and spin down.
4. Add 10 μL cDNA (Focus panel consisting of 2x 96 assays).
5. Add 10 μL PCR Master mix: cDNA mix to each well3).


See tip 8
Protocol C.
2) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.
40

Step 8 Spin plate briefly in a plate centrifuge (1500g for 1 minute), to collect
Spin plate the sample.
Step 9 Perform real-time PCR amplification followed by melting curve analysis

Real-time PCR according to Table 20.
amplification
Process step Settings, LC480 Settings, other
instrument5) instruments 4)
Polymerase 95˚C, 10 min 95˚C, 10 min
Activation/Denaturation
cycles at cycles at
95˚C, 10 s 95˚C, 10 s
60˚C, 1 min, 60˚C, 1 min,
ramp-rate ramp-rate
1.6˚C/s6) 1.6˚C/s6)
4) If using a 96-well cycler with a minimum recommended volume of 20 μL (like some ABI instruments), then use 10 μL reaction volume
Protocol C.
5) Five additional amplification cycles is required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40.
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier. Note:
The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
41
PCR instrument). If you are using an ABI instrument, please note that it
is not recommended to use auto Ct settings. Furthermore, if the data is
to be analyzed using Exiqon GenEx, the experiment must be set up as
an AQ experiment, not RQ. For a guide on how to set manual baseline
and threshold, refer to Tip 10, page 56 in the tips section. If you are
using a Roche LC480 instrument, we recommend analysis using the
2nd derivative method.
Please refer to our data analysis guide for recommendations.
See tip 11
Protocol C.
42
Protocol D - Pick-&-Mix microRNA PCR

Panels
using the Pick-&-Mix microRNA PCR Panel, 96-well plates (product number 203893-
203897) and 384-well plates (product number 203818-203819).
If working with serum plasma samples or biofluid samples, please refer to the specific

• Swing bucket centrifuge for 96/384-well plates.
Checklist:
• ROX: The ExiLENT SYBR® Green master mix does not include the ROX passive reference dye. Please
follow instrument manufactures recommendations
• ABI instruments: The use of manual background and threshold settings is necessary for obtaining
correct PCR data. Furthermore, if the data is to be analyzed using GenEx, the experiment must be
set up as an AQ experiment, not RQ. Make sure to have the optimal settings by downloading the Protocol D. Pick-&-Mix
microRNA PCR Panels
instrument settings file at www.exiqon.com/sds.

43
Workflow for Pick-&-Mix PCR plates (per sample)



- Add cDNA:PCR Master mix to primer
sets replicates (indicated by colored boxes)
Protocol D. Pick-&-Mix
microRNA PCR Panels
4

1
0
-1
miR-21
let-7a
stroma
44
Pick-&-Mix microRNA PCR Panel layouts 22 microRNAs x 16 samples

Overview of the pre-defined plate layouts A
1
1 2
2 3
3
4
4
5
5
6
6
7
7
8
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
available for 96-well and 384-well plates of the B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
Pick-&-Mix Panel. Wells in dark gray and light E

F
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
CP
CP
IPC
IPC
gray are pre-occupied by interplate-calibrators G

H
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
CP
CP
IPC
IPC
I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
(UniSp3 IPC) and RNA spike-in controls J 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
K 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
(UniSp6), respectively. L
M
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
CP
CP
IPC
IPC
N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC
10 microRNAs x 8 samples 46 microRNAs x 8 samples

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 2 3 4 5 6 7 8 9 10 11 12 A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP
B 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
A 1 2 3 4 5 6 7 8 9 10 CP IPC
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP
B 1 2 3 4 5 6 7 8 9 10 CP IPC D 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP
C 1 2 3 4 5 6 7 8 9 10 CP IPC F 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP
D 1 2 3 4 5 6 7 8 9 10 CP IPC H 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
I CP
E
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1 2 3 4 5 6 7 8 9 10 CP IPC
J 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
F 1 2 3 4 5 6 7 8 9 10 CP IPC K 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP
L 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
G 1 2 3 4 5 6 7 8 9 10 CP IPC M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP
N 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
H 1 2 3 4 5 6 7 8 9 10 CP IPC O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP
P 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 IPC
22 microRNAs x 4 samples 94 microRNAs x 4 samples

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 2 3 4 5 6 7 8 9 10 11 12 A 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12
B 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12
A 1 2 3 4 5 6 7 8 9 10 11 IPC C 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24
D 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24
B 13 14 15 16 17 18 19 20 21 22 23 CP
E 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 CP CP 36 36
C 1 2 3 4 5 6 7 8 9 10 11 IPC F 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 CP CP 36 36
G 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48
D 13 14 15 16 17 18 19 20 21 22 23 CP H 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48
I 49 49 50 50 51 51 52 52 53 53 54 54 55 55 56 56 57 57 58 58 59 59 60 60
E 1 2 3 4 5 6 7 8 9 10 11 IPC J 49 49 50 50 51 51 52 52 53 53 54 54 55 55 56 56 57 57 58 58 59 59 60 60
K
F
61 61 62 62 63 63 64 64 65 65 66 66 67 67 68 68 69 69 70 70 IPC IPC 72 72
13 14 15 16 17 18 19 20 21 22 23 CP
L 61 61 62 62 63 63 64 64 65 65 66 67 67 67 68 68 69 69 70 70 IPC IPC 72 72
G 1 2 3 4 5 6 7 8 9 10 11 IPC M 73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84
N
microRNA PCR Panels
73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84
H 13 14 15 16 17 18 19 20 21 22 23 CP O 85 85 86 86 87 87 88 88 89 89 90 90 91 91 92 92 93 93 94 94 95 95 96 96
P 85 85 86 86 87 87 88 88 89 89 90 90 91 91 92 92 93 93 94 94 95 95 96 96
380 microRNAs x 1 sample

92 microRNAs x 1 sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 2 3 4 5 6 7 8 9 10 11 12 B 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
A 1 2 3 4 5 6 7 8 9 10 11 IPC C 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
D 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 IPC
B 13 14 15 16 17 18 19 20 21 22 23 CP E 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
F 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144
C 25 26 27 28 29 30 31 32 33 34 35 IPC
G 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168
H
D 37 38 39 40 41 42 43 44 45 46 47 IPC
169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 IPC
I 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216
E 49 50 51 52 53 54 55 56 57 58 59 60 J 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
K 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264
F 61 62 63 64 65 66 67 68 69 70 71 72 L 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 CP
M
G
289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312
73 74 75 76 77 78 79 80 81 82 83 84
N 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336
H 85 86 87 88 89 90 91 92 93 94 95 96 O 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360
P 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 IPC
45
Protocol
Step 1 Adjust each of the template RNA samples to a concentration of 5 ng/

Dilute template RNA μL using nuclease free water.
microRNA PCR Panels
46
Step 3 When performing first-strand cDNA synthesis on multiple RNA

Combine reagents samples, it is recommended to prepare an RT working solution of
according to Table 21 the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the
proportions indicated in the first four lines of Table 21).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Reagent Volume (μL) Volume (μL)

< 100 miRNA > 100 miRNA
analyzed per analyzed per
sample sample
5x Reaction buffer 2 4
Nuclease-free water 4.5 9
Enzyme mix 1 2
Synthetic RNA spike ins, 0.5 1
optional replace with H2O
if omitted
Template total RNA 2 4
(5ng/μL)
Total volume 10 20
Table 21. Reverse transcription reaction setup per sample1)
The amount of 100x fold diluted cDNA needed for the different
Pick-&-Mix layouts can be seen in Table 22 – Step 7. Protocol D. Pick-&-Mix
microRNA PCR Panels
Mix and spin reagents reagents are thoroughly mixed. After mixing, spin down.
1) The amount of cDNA required per sample depends on the number of microRNAs analyzed per sample. The volumes suggested here
provides excess amount of cDNA, which ensures the highest possible reproducibility because these volumes can be pipetted with
great accuracy.
47

Incubate and • Heat-inactivate the reverse transcriptase
heat inactivate2) for 5 min at 95˚C.
qPCR protocol:
Prepare reagents on ice and thaw for 15-20 min. Protect the PCR Master mix vials from
for real-time PCR light. Immediately before use, mix the PCR Master mix by pipetting up
down.
2) Although not recommended, the protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks
(optional store at 4˚C for up to 4 days). It is recommended that synthesized cDNA be stored in “low-nucleic acid binding” tubes or plates.
microRNA PCR Panels
48
Step 7 Dilute the cDNA from the RT reactions to give a final 100x dilution.
Dilute cDNA template Suggested cDNA dilution procedures is shown in Table 22 along with
100x in nuclease required volumes of diluted cDNA for the different Pick-&-Mix pre-
free water3) defined layouts. It is receommeded that “low-nucleic acid binding”.
For fully customized layouts dilutions may be adjusted to the specific
replicate scheme. tubes or plates are used. It is not recommended to
store the 1:100 dilution of cDNA.
Pick-&-Mix plate Suggested Volume (μL) of

configuration cDNA dilution diluted cDNA
procedure needed for each
cDNA + nuclease Pick-&-Mix plate
free water (μL)
8x10 (12) 2 + 198 60
4x22 (24) 2 + 198 120
1x92 (96) 5 + 495 480
16x22 (24) 2 + 198 120
8x46 (48) 3 + 297 240
4x94 (96) 5 + 495 480
1x380 (384) 20 + 1980 1920
Table 22. Amount of 100x diluted cDNA needed in

Pick-&-Mix plates
Numbers in parenthesis designate total number of assays/sample,

including controls and inter-plate calibrators. Loss from pipetting is
not included in the volumes. listed in the right column (diluted cDNA
microRNA PCR Panels
needed).

recommendations when deciding how much ROX to use, see Tip 8.
See tip 8
3) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.
49

Step 8 The following procedure is recommended:

Combine cDNA and 1. Before removing the plate seal, briefly spin down the plate(s) in a
PCR Master mix 1:1 plate centrifuge.
and add to PCR plates 2. Combine 2x PCR Master mix and 100x diluted cDNA 1:1 (e.g. 500 μL
2x PCR master mix and 500 μL diluted cDNA).
Mix thoroughly 3. Mix gently by inverting the tube, spin down.
4. Add 10 μL PCR Master mix:
cDNA mix to each well.4)

Step 9 Spin plate briefly in a plate centrifuge (1500g for 1 minute), to remove
Spin plate air bubbles.
Step 10 Perform real-time PCR amplification followed by melting curve

Real-time analysis according to Table 23.
PCR amplification
Process step Settings, LC480 Settings, other
instrument5) instruments6)
microRNA PCR Panels

Denaturation
cycles at cycles at
95˚C, 10 s 95˚C, 10 s
60˚C, 1 min, 60˚C, 1 min,
ramp-rate ramp-rate
1.6˚C/s7) 1.6˚C/s7)
50
PCR instrument). If you are using an ABI instrument, please note that
it is not recommended to use auto Ct settings. For a guide on how to
set manual baseline and threshold, refer to Tip 10, page 56 in the tips
section. Furthermore, if the data is to be analyzed using Exiqon GenEx,
the experiment must be set up as an AQ experiment, not RQ.
For tips on normalization, please see Tip 11, page 58. If you are using
a Roche LC480 instrument, we recommend analysis using the 2nd
derivative method. We recommend performing normalization and
further data analysis with the Exiqon GenEx qPCR analysis software
(www.exiqon.com/mirna-pcr-analysis). Please refer to our data
analysis guide for recommendations.
See tip 11

5) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40.
6) If using a 96-well cycler with a minimum recommended volume of 20 μL (like some ABI instruments), then use 10 μL reaction volume
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier.
Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
microRNA PCR Panels
51
Tips to protocol
Tip 1. RNA extraction and template preparation
Purification and preparation of total RNA that includes small RNAs (<200 nt) from a
biological sample is the first critical step for a successful expression profiling analysis
of microRNAs. Therefore, the method used for RNA sample preparation is critical to the
success of the experiment.
The following points should be considered before starting the experiment:

• We recommend using the miRCURY™ RNA Isolation kit for isolation of RNA that contains
the small RNA fraction. If not using one of the miRCURY™ RNA Isolation kits, it is
important that the method used to isolate RNA from a sample should give a quantitative
recovery of small RNAs and should not result in a substantial loss of the small RNA
fraction. This also applies when using commercially available kits. Make sure the total
RNA preparation is guaranteed to contain microRNA.
• If commercially available purified RNA is used, it is important to make sure that the
RNA is guaranteed to contain small RNAs and is representative of the microRNAs in the
species and/or tissue from which it was isolated.
• The comparison of samples prepared using different RNA isolation methods is not
recommended. However, if this is necessary, it is recommended to include the measure
of a reference small RNA which has a consistent and unvaried expression level in order to
allow for comparison of microRNA levels in the different sample preparations.
• The isolation of RNA and the reaction steps preceding real-time PCR should be performed
in rooms separate from where real-time PCR experiments will take place in order to avoid
contamination with amplicon.
• It is recommended that the integrity of isolated RNA be assessed before proceeding with
quantitative real-time PCR experiments. This may be performed either on the Agilent
2100 Bioanalyzer or by denaturing gel electrophoresis.
• If necessary, treat RNA preparations with DNases to remove contaminating DNA that may
interfere with the real-time PCR results.
• Purified total RNA should be dissolved in nuclease-free water at a stock concentration of
at least 1 μg/μL. See recommendations for storage conditions in Tip 3.
52
Tip 2. Guidelines for serum, plasma and biofluid samples
Plasma and serum are essentially cell free liquid samples. This means that only circulating
RNA is extracted from these sample types, resulting in low total RNA concentrations,
even if the microRNA fraction is readily detectable. The result of this is that measuring
correct RNA concentrations is difficult, and that there is a high risk of increased loss during
extraction. For this reason, we recommend using RNA amounts based on starting volume
rather than RNA quantity. Comprehensive guidelines for microRNA profiling in blood serum/
plasma can be downloaded at www.exiqon.com/serum-plasma-guidelines.
Tip 3. General guidelines for handling and storage of RNA
The following precautions should be taken to prevent RNase contamination and degradation
of the RNA sample and reagents:
• Always wear disposable gloves.
• Use nuclease-free, low nucleic acid binding plasticware and filter barrier pipette tips.
• Keep tubes capped when possible. Always spin tubes before opening.
• For long-time storage, RNA may be stored at -80°C. Avoid repeated freezing and thawing
cycles.
Tip 4. Good PCR laboratory practice
To reduce the risk of contaminating PCRs with “old” PCR amplicons, and consequently
obtain false results:
• Always wear a clean lab coat. Use separate lab coats for RNA sample preparation, cDNA
synthesis and when setting up PCR reactions or handling PCR products.
• Change gloves oen, especially whenever you suspect they may have been contaminated.
• Establish and maintain designated areas for PCR set-up, PCR amplification, and gel
electrophoresis of PCR products.
• Never bring amplified PCR products into the PCR set-up area.
• Spin down all reaction and sample tubes before opening. Open and close all reagent and
sample tubes carefully, trying not to splash or spray PCR samples.
• Keep reactions and components capped whenever possible.
• Use filter barrier pipette tips to avoid aerosol-mediated contamination of your pipetting
device.
• Clean laboratory benches and equipment regularly.
53
Tip 5. Keep reagents and reactions cool at all times
In order to ensure optimal performance of the miRCURY LNA™ Universal RT microRNA

PCR system it is important that the reagents and reactions are kept on ice (or at 4°C) as
much as possible during the protocol (apart from reaction steps specifically involving raised
temperatures).
Tip 6. First strand cDNA synthesis
Store cDNA samples in nuclease-free low nucleic acid-binding micro centrifuge tubes, e.g.
Eppendorf DNA LoBind tubes.
Tip 7. Recommended controls
The following controls are recommended and should be included in the experimental set-up:
• Reverse transcription/no enzyme controls, i.e. first-strand cDNA synthesis reactions
performed without the Enzyme mix. If a PCR product is amplified from this control
reaction it indicates genomic DNA or PCR product contamination of the template RNA.
• Non-template controls in the real-time PCR amplification, i.e. real-time PCR
reactions performed without any cDNA template. This control will reveal PCR product
contamination of the reaction.
• Blank purification or carrier only, i.e. when purifying RNA in the presence of carrier RNA
(such as for serum and plasma samples). This control will reveal any non-specific signals
originating from the carrier RNA alone.
Tip 8. Passive reference dye (ROX)
Many real-time PCR instruments will only produce reliable results when a passive
reference dye such as ROX is added to the PCR reaction. The reference dye is used to
normalize signals from individual PCR wells in order to enable comparison of real-time PCR
amplification signals across an entire PCR-plate.
It is recommended to determine whether your real-time PCR instrument has this type
of requirement. The amount of ROX to include in the PCR reaction depends on the
requirements of the real-time PCR instrument and must be adjusted accordingly. Please
follow the supplier’s instructions for preparation and concentrations of ROX solutions.
Typically, real-time PCR instruments that allow excitation at individual wavelengths for
54
individual dyes (most filter wheel based instruments) require less ROX than instruments that
use a single excitation wavelength for all fluorophores (most laser based instruments use
excitation at 488 nm). It is important to note that excessive amounts of ROX may inhibit the PCR
reaction. It may be recommended to perform a titration of ROX amounts in order to determine
the optimal concentration for a particular instrument-system combination. It is possible to use
the spike-in template (provided in the Universal cDNA synthesis kit II) and the Control primers
(provided in the ExiLENT SYBR® Green master mix kit) to perform such titrations.
Recommended ROX concentrations are found here:
Instrument ROX concentration

ABI 7900HT 300-500 nM
ABI 7900HT FAST 300-500 nM
ABI Viia7 30-50 nM
ABI Viia7 FAST 30-50 nM
ABI 7500 FAST 30-50 nM
ABI StepOnePlus 300-500 nM
ABI 7300 300-500 nM
ABI 7500 30-50 nM
ABI 7700 300-500 nM
ABI 7000 300-500 nM
Table 24.
Tip 9. Inter-plate calibration
When setting up large scale experiments with several PCR plates involved, individual run
differences may be observed. One way of avoiding these having an effect on the results
is setting up the experiment in such a way that all samples for one gene are run on the
same plate. However, this is not always feasible. When Cq values for one gene have to be
compared across plates, it is recommended to employ an inter-plate calibrator.
An inter-plate calibrator is a template-assay combination which is the same in all plates,

and always located in the same well across different plates. This can then be used to
calibrate all plates to give the same Cq value for the calibrator, thereby reducing run-to-run
variance. Please note that all panels contain inter-plate calibrators.
55
Tip 10. Guidelines for real-time PCR data collection using ABI instruments
On cyclers using baseline and threshold values for Cq (Ct) calculations, such as ABI 7900HT,
it is important that the proper settings are used. Use of the automatic function of the
soware for these settings does not seem to produce optimal results for SYBR® Green
based assays. Oen the baseline is set erroneously on non-detected assays, and this in turn
gives false positives, therefore do not use automatic settings. Another issue to consider
when using automatic settings is that the settings may differ between plates resulting in
data that cannot be compared directly. Inter-plate calibration may not fully resolve this
issue, since each assay has a separately calculated baseline and threshold. Instead, both
threshold and baseline should be set manually, applying the same settings for all assays on
the plate.
The following principles should be applied to manual baseline and threshold settings:
Baseline:
The baseline should be calculated in the cycle interval before the amplification takes off
(see Figure 3).
Threshold:
The threshold should then be set with the Y-axis in log scale where all assays are in the log
linear phase, and the threshold above background for all assays (see Figure 3).
Note: The optimal threshold value may vary between individual machines and experiments.
Important note
If ROX passive reference dye has not been used in the PCR reactions, make sure the
SDS soware is set-up without reference dye correction.
56
Baseline setting
Threshold above
background
Amplification
take-off
Baseline
interval
Figure 5.
57
Tip 11. Quick guide to normalization of microRNA qPCR experiments
The purpose of normalization is to remove technical and biological variation between

samples that is not related to the biological changes under investigation. Proper
normalization is critical for the correct analysis and interpretation of results from real-
time PCR experiments. The most commonly used option for normalization is to use stably
expressed reference genes.
In general it is recommended to test several endogenous control candidates (reference

genes) before setting up the actual microRNA expression analysis. These candidates should
be chosen among genes that can be expected to be stably expressed over the whole range
of samples being investigated. They can be stably expressed small non-coding RNA, or
stably expressed microRNAs, and chosen based on literature or pre-existing data (e.g.
microarray analysis or qPCR panel screening).
Exiqon offers primer sets for a number of different small RNAs which tend to be stably
expressed, and are therefore oen good candidates for reference genes. It’s important to
keep in mind that in spite of being small non-coding RNAs, most of these are significantly
larger than microRNA and therefore may have different extraction efficiency and stability.
U6 is one such reference gene which is oen used. However, U6 is significantly larger than
microRNAs and has a different sub-cellular distribution. The existence of several different
isoforms also makes it a suboptimal reference gene. 5S ribosomal RNA is another popular
option, but this RNA has a much higher expression level than most microRNAs, and is oen
found as a PCR contaminant.
If working with blood serum or plasma, please note that only circulating RNA are present.
In this case the small non-coding RNAs (5S, U6, SNORs etc) are not good candidates for
reference genes, since they are most probably not present in the sample.
58
Using stably expressed microRNAs as reference genes offers several advantages such as
equal size, extraction efficiency and stability, as well as having expression levels within a
similar range of the target microRNAs. Several candidates can be found in the literature,
including miR-191-5p, miR-103a-3p, let-7a-5p, and miR-16-5p. Microarray or qPCR panel
screening data may also be used when choosing candidate reference genes.
All reference gene candidates should be empirically validated for each study. A number
of different soware packages exist for evaluating the optimal nature and number of
endogenous controls, and for applying multiple endogenous controls for normalizing
target expression. One such option is the GenEx soware from MulitD, sold through Exiqon
with a special application for Exiqon PCR panels. GenEx incorporates both GeNorm and
Normfinder for finding the optimal reference genes, and is easy and intuitive to use for the
actual normalization.
An alternative option for normalization of data from panels (profiling a high number of
microRNAs) is to normalize against the global mean; that is, the average of all expressed
microRNAs This can be a good option in samples with a high call-rate (expressed
microRNAs), but should be used with caution in samples with low call-rates. It is also not a
good option in samples for which the general microRNA expression level is changed.
For further information on normalization and references, we recommend our data-analysis

guide available online as well as the guide to microRNA normalization from
www.gene-quantification.de.
Tip 12. Melting curves
Melting curves are a good tool for evaluating the specificity of the assay. When setting up
the melt curve temperature gradient on the instrument, it is recommended to start at 60˚C.
59
Troubleshooting guide
Problem Suggestion
PCR signal in samples This typically indicates contamination of the template RNA with
amplified from first- genomic DNA. Perform DNase treatment of the RNA sample. If this
strand synthesis does not solve the problem RNA samples or other reagent may be
reactions performed contaminated with PCR products.
without reverse
transcriptase
PCR signal in This typically indicates contamination of the cDNA template or PCR
no-template reagents with amplified PCR product.
PCR reaction
Exposing the reactions to elevated temperatures (i.e. room
temperature) during any part of the protocol increases the risk of
background signals. It is important that the reagents and assembled
reactions are kept cool (on ice or 4°C) at all times (see Tip 4,
page 53 for details).
Generated signals • On some real-time PCR cyclers, gain-settings are adjustable.
are weak Make sure the gain settings of your real-time PCR cycler have been
set to accommodate the signals generated from the specific assay.
• RNA samples may contain PCR inhibitors. Further purification or
an alternative RNA extraction method may be necessary. Check
positive controls.
No fluorescent signal is Confirm that you have a PCR product by running an aliquot of your
detected during the PCR PCR reaction on an agarose gel.
No fluorescent signal • Check that the filter in the real-time PCR cycler was set to either
detected during the PCR, SYBR® Green or FAM/FITC.
but a PCR amplicon can • Check that the optical read is at the correct step of the real-time
be detected by agarose PCR cycles.
gel electrophoresis • Adjust the baseline in the real-time PCR cycler software.
60
FAQs
What kind of real-time PCR instruments is miRCURY LNA™ Universal RT microRNA
PCR compatible with?
miRCURY LNA™ Universal RT microRNA PCR is compatible with all instruments capable of
reading green fluorophores such as fluorescein/FITC/FAM and SYBR® Green. The system has
been tested and found to work on real-time PCR instruments from several leading suppliers
of this type of instrument.
What kind of settings should I use on my real-time PCR instrument?

If your real-time PCR instrument supports fluorophores such as fluorescein/FITC/FAM or
SYBR® Green your instrument must be set to detect these fluorophores.
Is miRCURY LNA™ Universal RT microRNA PCR compatible with other SYBR® Green
master mixes?
We do not recommend using other SYBR® Green master mixes for real-time PCR analysis
with the LNA™ PCR primer sets or Ready-to-Use panels. The primer sets have been
optimized and validated using the miRCURY LNA™ ExiLENT SYBR® Green master mix and
the performance of the primer sets will be compromised by using a different master mix
(which may contain different salt and/or enzyme concentrations).
My RNA is already enriched for microRNA, how much should I use in the real-time PCR
experiments?
miRCURY LNA™ Universal RT microRNA PCR is developed for use on total RNA and we do
not recommend enriching for small RNAs. Samples of enriched microRNAs are difficult to
quantitate accurately making it very tricky to ensure the same amount of sample is added
to each reaction. If necessary, a total RNA equivalent should be used for the enriched
sample, e.g. use a proportional amount of enriched sample resulting from 20 ng of total
RNA. It may be necessary to try a couple of different amounts of enriched sample to ensure
that the results fall within the linear range of the assay.
61
What is the recommended experimental set up for Universal RT microRNA real-time PCR?
It is generally accepted that the reverse transcription (RT) reaction gives rise to more
variation than the PCR reaction. It is therefore advisable to perform replicate RT reactions,
ideally 3 separate reactions with 1-2 PCR reactions for each RT. It is further recommended
to always include at least three biological replicates (separate RNA extractions) of
each sample type in order to allow statistical analysis of the results. If small changes in
microRNA expression are expected, it may be necessary to include more replicates to
ensure a signifi cant result. In general it is recommended that replicates should be included
at any stage during sample procurement, processing, RNA isolation, etc. that could give rise
to variation between samples.
A tech note on guidelines for setting up microRNA qPCR experiments can be downloaded at
www.exiqon.com/miRNA-qPCR-guidelines
Additional information is available at www.exiqon.com/mirna-pcr
62
Related products
Exiqon offers a broad variety of tools enabling new discoveries concerning the expression,
function and spatial distribution of microRNAs:
miRCURY LNA™ microRNA Array, microarray kit

Pre-printed miRCURY LNA™ microRNA Array microarray slides, available for hsa, mmu
& rno and other species. Kit includes hybridization and wash buffers as well as synthetic
spike-in microRNAs.
miRCURY LNA™ microRNA Power labeling kit

For fluorescent labeling of microRNAs from total RNA samples ready for array
hybridization.
miRCURY LNA™ microRNA Array, ready-to-spot probe set

Ready-to-spot oligos for direct printing of arrays, or coupling in bead-based applications.
miRCURY LNA™ microRNA Detection Probes

For in situ hybridization and northern blotting of all annotated microRNAs.
miRCURY LNA™ microRNA ISH Optimization kit (FFPE)

Complete kit with control probes and hybridization buffer for easy set up of microRNA
in situ hybridization.
miRCURY LNA™ microRNA Inhibitors, Power Inhibitors and Family Inhibitors

Unravel the function of microRNAs by microRNA inhibition. Sophisticated LNA™ design
ensures potent inhibition of all microRNAs regardless of their GC content. Chemically
modified, highly stable Power Inhibitors for unrivalled potency. Available for in vitro and
in vivo studies.
miRCURY LNA™ microRNA Inhibitor Library

For genome-wide high throughput screening of microRNA function.
miRCURY LNA™ Target Site Blockers

For inhibition of microRNA binding to a specific mRNA target.
63
Notice to purchaser
Exiqon, LNA and miRCURY are registered trademarks of Exiqon A/S, Vedbaek, Denmark. SYBR Green is a licensed
trademark of Invitrogen. All other trademarks are the property of their respective owners.
Locked-nucleic Acids (LNAs™) are protected by US Pat No. 6,268,490, US Pat No. 6,770,748, US Pat No. 6,639,059,
US Pat No. 6,734,291 and other applications and patents owned or licensed by Exiqon A/S. Products are provided
to buyers for research use only. The products in their original or any modified form may be used only for the buyer’s
internal research purposes and not for commercial, diagnostic, therapeutic, or other use, including contract research.
The buyer may not provide products to third parties in their original or any modified form. The purchase of products
does not include or carry an implied right or license for the buyer to use such products in their original or any modified
form in the provision of services to third parties, and a license must be obtained directly from Exiqon A/S for such
uses.
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside
the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, non transferable immunity from
suit under the foregoing patent claims for using only this amount of product solely in Contract Research, including
reporting results of purchaser’s activities for a fee or other commercial consideration, and also for the purchaser’s
own internal research. No right under any other patent claim is conveyed expressly, by implication, or by estoppel.
Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Furthermore, this product is provided under an agreement between Molecular Probes, Inc., a wholly owned subsidiary
of Invitrogen Corporation, and EXIQON and the manufacture, use, sale or import of this product is subject to one or
more U.S. Patents and corresponding international equivalents. The purchase of this product conveys to the buyer
the non-transferable right to use the purchased amount of the product and components of the product in research
conducted by the buyer, where such research does not include testing, analysis or screening services for any third
party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its
components or (c) materials made using this product or its components to a third party or otherwise use this product
or its components or materials made using this product or its components for Commercial Purposes. Commercial
Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product
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information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc.,
Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300, Fax: (541) 335-0354.
Further, the purchase of this product includes a limited, non-transferable license under specific claims of U.S.
Patent Nos. 6,174,670 and 6,569,627, owned by the University of Utah Research Foundation and licensed to Roche
Diagnostics GmbH and Idaho Technology, Inc., to use only the enclosed amount of product according to the specified
protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any
claim of U.S. Patent Nos. 6,174,670 and 6,569,627, other than for the amount of product contained herein.
For life science research use only. Not for use in diagnostic procedures.
64
Notes
65
Outside North America
Exiqon A/S
Skelstedet 16
DK-2950 Vedbaek, Denmark
Phone +45 45 650 929
Fax +45 45 661 888
North America
Exiqon Inc.
13-0099 - 920003 - v6.2 - 07/2016 - NORMAL
12 Gill Street, Suite 1650

Woburn, MA 01801, United States
Phone (781) 376 4150
Fax (781) 376 4152
exiqon.com

Universal RT MicroRNA PCR Manual

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miRCURY LNA™ Universal RT

Universal cDNA synthesis kit II RNA Spike-in kit

Description page 6, Description page 9, Description page 7,

Universal cDNA synthesis kit II, 8-64 rxns (product # 203301)

Contents Amount supplied

miRCURY LNA™ Universal RT microRNA PCR, Starter Kit (product # 203351)

Contents Amount supplied

Contents Amount supplied

Contents Amount supplied

Contents Amount supplied

II. Primer Sets and Panels

MicroRNA LNA™ PCR primer set (product # 204000-206xxx and 2100000-21xxxxx)

Reference gene primer set (product # varies)

Pre-deﬁned microRNA PCR Panels (product # varies)

Contents Human miRNome panel I and II, V4

Contents Mouse&Rat miRNome panel I and II, V4

Contents Serum/Plasma Focus microRNA PCR panel, V4

96-well (2 plates) 384-well plate (2 panels per plate)

Contents Cancer Focus microRNA PCR panel, V4

96-well (1 plates) 384-well plate (4 panels per plate)

1) Please go to www.exiqon.com/mirna-pcr to download plate layout ﬁles.

Pick-&-Mix microRNA PCR Panel, (product # 203893-203897 and 203818-203819)

96-well PCR plates1) 384-well PCR plates1)

1) Please go to www.exiqon.com/pick-and-mix to conﬁgure a Pick-&-Mix microRNA PCR Panel.

Additional required materials

Reagents not supplied

Materials and Equipment not supplied

Optional but recommended product

Recommended accompanying products

Exiqon GenEx6 Industrial - Exiqon version of GenEx,

Exiqon GenEx6 Academic - Exiqon version of GenEx,

miRCURY™ RNA Isolation Kit – Cell & Plant

miRCURY™ RNA Isolation Kit – Tissue

miRCURY™ RNA Isolation Kit – Bioﬂuids

Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System.

Step 1: First-strand synthesis (RT)

Step 2: Real-time PCR ampliﬁcation

miR-speciﬁc reverse primer

Reference assays and reference candidates

Plate 1 Plate 2 Plate 3

Table 13. Example of inter-plate calibration (IPC)

RNA spike-ins (synthetic control templates)

If UniSp6 is to be used alone:

An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This is

Comes with Assays available as

UniSp2 UniSp4 UniSp5 cel-miR-39-3p UniSp6 UniSp3 miR-A miR-X

Before starting the Experiment

RNA work requires speciﬁc handling and precautions to prevent RNase

Normalization – when running individual assays or when conﬁguring a Pick-&-Mix

Serum plasma or other bioﬂuids

Use standard printed manual or download from

Protocol A - Individual assays

Additional required materials:

Workﬂow for individual primer sets (per sample)

Phase I: Prepare RNA sample

Phase II: cDNA synthesis

Phase III: real-time PCR amplification

LNATM primer sets

2 See data analysis guide online

First strand synthesis

Step 1 Adjust each of the template RNA samples to a concentration of

Step 3 If performing first-strand cDNA synthesis on multiple RNA samples, it

Table 14. Reverse transcription reaction setup

Step 5 • Incubate for 60 min at 42˚C.