For general laboratory use.

Not for use in diagnostic

procedures. FOR IN VITRO USE ONLY.

High Pure RNA Isolation Kit

For small-scale (mini) preparations of RNA
Cat. No. 11 828 665 001
Kit for 50 reactions Version December 2008
Store at
15 to
25°C

1. What this Product Does Additional Equipment and Reagents Required

• Absolute ethanol
Number of Tests
• Standard tabletop microcentrifuge capable of 13,000 × g centrifu-
The content of 1 kit is sufficient for 50 isolations of total RNA. gal force
• Microcentrifuge tubes, 1.5 ml, sterile
Kit Contents
for the isolation of total RNA from human blood:
N All solutions are clear, and should not be used when precipitates
have formed. Warm the solutions at +15 to +25°C or in a 37°C • Red Blood Cell Lysis Buffer*
waterbath until the precipitates have dissolved. • PBS*
for the isolation of total RNA from yeast:
Vial/Cap Label Contents / Function
• Lyticase (0.5 mg/ml)
1 Lysis/-Binding • 25 ml • PBS*
green Buffer • [4.5 M guanidine-HCl, 50 mM Tris-
for the isolation of total RNA from bacteria:
HCl, 30% Triton X-100 (w/v), pH 6.6
(25°C)] • Lysozyme* (stock solution 50 mg/ml, store aliquoted at 15 to
25°C).
2 DNase I, recombi- • 10 KU lyophilized DNase I
• 10 mM Tris, pH 8.0.
nant, lyophilizate • Resuspend in 0.55 ml Elution Buffer
3 DNase Incubation • 10 ml Application
white Buffer • [1 M NaCl, 20 mM Tris-HCl and 10 The High Pure RNA Isolation Kit is designed for the purification of total
mM MnCl2, pH 7.0 (25°C)] RNA from cultured cells. Other sample materials like blood, yeast and
bacteria require an additional specific pre-lysis treatment, which is
4 Wash Buffer I • 33 ml (add 20 ml ethanol p.a. before described in the protocol section.
black first use)
Due to the integrated DNase digestion step, contamination of the iso-
• [5 M guanidine hydrochloride and lated RNA with residual genomic DNA is mostly avoided. In addition,
20 mM Tris-HCl, pH 6.6 (25° C); final RNA is suited for other techniques like northern blotting, RNase pro-
concentrations after addition of 20 tection and primer extension. Up to 24 samples can be processed
ml ethanol p.a.] simultaneously in approx. 1 hour. Thus, the purification procedure is
5 Wash Buffer II • 10 ml (add 40 ml ethanol p.a. before less time consuming compared with alternative methods which
blue first use) require extraction with organic solutions, RNA precipitation or ultra-
• [20 mM NaCl, 2 mM Tris-HCl, pH 7.5 centrifugation.
(25°C); final concentrations after
addition of 40 ml ethanol p.a.] 2. How to Use this Product
6 Elution Buffer • 30 ml 2.1 Before You Begin
colorless • Nuclease-free, sterile, double dis- Precautions
tilled water N Lysis/-Binding Buffer and Wash Buffer I contain guanidine hydro-
7 High Pure Filter One bag with 50 polypropylene tubes chloride which is a chemical hazard and irritant.
Tubes with two layers of glass fiber fleece, • Avoid contact of the buffers with skin, eyes, or mucous membranes.
for uptake of up to 700 l sample vol- If contact does occur, immediately wash with large amounts of
ume water. Burns can occur if left untreated. If the reagent spills, dilute
8 Collection Tubes One bag with 50 polypropylene tubes with water before wiping dry.
(2 ml) • Always store and use the buffers away from food for humans and
animals.
Storage and Stability • Always wear gloves, and follow standard safety precautions during
The High Pure RNA Isolation Kit components must be stored at +15 to handling.
+25°C. Kit components are guaranteed to be stable through the expi-
ration date printed on the label. Handling Requirements
N Please note, that improper storage at +2 to +8°C (refrigerator) or • Exercise the normal precautions required for handling all laboratory
15 to 25°C (freezer) will adversely impact nucleic acid purifica- reagents.
tion when precipitates form in the solutions. • The use of sterile, disposable polypropylene tubes and tips is rec-
Therefore, High Pure isolation kits are always shipped at +15 to ommended in order to avoid RNase contamination. Wear gloves
+25°C. during the assay.
Reconstituted DNase solution has to be stored in aliquots. Aliquots • Do not pool reagents from different lots or from different bottles of
stored at –15 to –25°C are stable for 1 year. the same lot. Immediately after usage, close all bottles in order to
avoid leakage, varying buffer-concentrations or buffer conditions.
After first opening store all bottles in an upright position.
1208.11824376001•
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Laboratory Procedures 2.2 Isolation of Total RNA from Cultured Cells
• All human sourced material and all resulting waste should be con- (suited for 1 × 106 cells)
sidered as potentially infectious.
쐃 Resuspend cells in 200 l PBS.
• Thoroughly clean and disinfect all work surfaces with disinfectants
recommended by the local authorities. 쐇 Add 400 l Lysis/-Binding Buffer (green cap) and vortex for 15 s.
• Do not eat, drink or smoke in the laboratory work area. 쐋 To transfer the sample to a High Pure Filter Tube:
• Do not pipette by mouth. • Insert one High Filter Tube in one Collection Tube.
• Wear protective disposable gloves, laboratory coats and eye protec- • Pipet entire sample into the upper reservoir of the Filter Tube
tion when handling specimens and kit reagents. (max. 700 l)
• Avoid microbial and nuclease contamination of reagents when 쐏 • Insert the entire High Pure Filter Tube assembly into a standard
removing aliquots from reagent bottles. tabletop centrifuge.
• The use of sterile disposable pipettes is recommended. • Centrifuge the tube assembly 15 s at 8,000 × g.
• Wash hands thoroughly after handling samples and test reagents. 쐄 After centrifugation:
• Remove the Filter Tube from the Collection Tube, discard the
Waste Handling flowthrough liquid, and again combine the Filter Tube and the
• Disposal of unused reagents and waste should occur in accordance used Collection Tube.
with country, federal state and local regulations. 쐂 After re-inserting the Filter Tube:
• Material Safety Data Sheets (MSDS) are available upon request • Pipette per sample 90 l DNase Incubation Buffer (white cap)
from the local Roche office. into a sterile reaction tube, add 10 l DNase I, mix and pipette
the solution on the glass filter fleece in the upper reservoir of
Sample Material the filter tube.
• 106 cultured cells • Incubate for 15 min at +15 to +25°C.
• 200 – 500 l human blood 쐆 • Add 500 l Wash Buffer I to the upper reservoir of the Filter
• 108 yeast cells Tube assembly and centrifuge 15 s at 8,000 × g.
• 109 bacterial cells (gram positive or gram negative) • Discard flowthrough and combine Filter Tube with the used
Collection Tube.
Preparation of Working Solutions 쐊 • Add 500 l Wash Buffer II (blue cap) to the upper reservoir of
Beside the ready-to-use solutions supplied with this kit, you will need the Filter Tube assembly and centrifuge 15 s at 8,000 × g.
to prepare the following working solution: • Discard flowthrough and combine Filter Tube with the used
Collection Tube.
Content Reconstitution/ Storage and For use in
Preparation Stability 쐎 Add 200 l Wash Buffer II (blue cap) to the upper reservoir of the
Filter Tube assembly and centrifuge for 2 min at maximum speed
DNase I Dissolve DNase I in Aliquot and store Removal of (approx. 13,000 × g) to remove any residual Wash Buffer.
(Vial 2) 0.55 ml Elution at –15 to –25° C. contaminat-
Buffer Stable for 12 ing DNA L The extra centrifugation time ensures removal of residual
months. Wash Buffer
Wash Add 20 ml absolute Store at +15 to Removal of 쐅 Discard the Collection Tube and insert the Filter Tube into a
Buffer I ethanol to Wash +25°C. Stable residual clean, sterile 1.5 ml microcentrifuge tube.
(Vial 4; Buffer I and mix through the expira- impurities 쐈 To elute the RNA:
black cap) well. tion date printed on • Add 50 – 100 l Elution Buffer to the upper reservoir of the Fil-
N Label and date kit label. ter Tube.
bottle accord- • Centrifuge the tube assembly for 1 min at 8,000 × g.
ingly after adding
ethanol. 쐉 The microcentrifuge tube now contains the eluted RNA.
Either use the eluted RNA directly in RT-PCR or store the eluted
Wash Add 40 ml absolute Store at +15 to Removal of RNA at –80°C for later analysis.
Buffer II ethanol to Wash +25°C. Stable residual
(Vial 5; Buffer II and mix through the expira- impurities 2.3 Isolation of Total RNA from Human Blood
blue cap) well. tion date printed on (suited for 200 – 500 ␮l whole blood)
N Label and date kit label. N Due to the modest content of RNA in leukocytes, it is recom-
bottle accord- mended, to use the isolated RNA exclusively in RT-PCR. Use
ingly after adding EDTA-preserved fresh whole blood. Erythrocytes are lysed by
ethanol. hypotonic lysis. We recommend the application of the Red Blood
Cell Lysis Buffer; this method is described in the following section.
쐃 Add 1 ml Red Blood Cell Lysis Buffer to a sterile 1.5 ml reaction
tube.
쐇 Add 500 l human whole blood and mix by inversion.
N Do not vortex.
쐋 Place the tube on a rocking platform or gyratory shaker for 10
min at +15 to +25° C.
L Alternatively, manually invert the sample periodically for 10
min.
쐏 Centrifuge for 5 min at 500 × g in a standard table top centrifuge.
쐄 • With a pipette, carefully remove and properly dispose of the
clear, red supernatant.
• Add 1 ml Red Blood Cell Lysis Buffer to the white pellet and
mix by “flicking” the tube until the pellet is resuspended.
N Do not vortex.

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쐂 • Centrifuge for 3 min at 500 × g. 4. Troubleshooting
• Carefully remove and properly dispose of the supernatant, par-
ticularly the red ring of blood cell debris that forms around the Possible Cause Recommendation
outer surface of the white pellet. Low nucleic Kit stored under Store kit at +15 to +25°C at all
쐆 Resuspend the white pellet in 200 l PBS and follow the protocol acid yield or non-optimal times upon arrival.
2.2 as described for cultured cells from step 2. purity conditions
Buffers or other • Store all buffers at +15 to +25°C.
2.4 Isolation of Total RNA from Yeast reagents were • Close all reagent bottles tightly
(suited for 1 x 108 cells) exposed to condi- after each use to preserve pH,
L It is recommended to harvest cells during the mid-log or late-log tions reducing their stability, and freedom from con-
phase of growth (OD600  2.0). The cell number can be counted in functionality. tamination.
a hemocytometer chamber or determined by measuring the opti- • Store the reconstituted Poly(A)
cal density at 600 nm in a spectral photometer. Use a dilution aliquoted at –15 to –25°C..
which gives a A600 of 0.1 – 0.15/ml (0.1 A600 correspond to approx.
2 × 106 cells.) No ethanol added 1. Add absolute ethanol to the buff-
to Wash Buffer ers before use.
쐃 • Collect the sample by centrifugation at 2,000 × g for 5 min in a 2. After adding ethanol, mix the
standard table top centrifuge. buffers well and store at +15 to
• Resuspend the pellet in 200 l PBS. +25°C.
3. Always mark Wash Buffer I and
쐇 Add 10 l Lyticase (0.5 mg/ml), incubate for 15 min at 30°C. Wash Buffer II vials to indicate
쐋 Follow protocol 2.2 as described for cultured cells from step 2. whether ethanol has been added
or not.
2.5 Isolation of Total RNA from Bacteria (gram positive Reagents and sam- Always mix the sample tube well
and gram negative) (suited for 1 x 109 cells) ples not completely after addition of each reagent.
mixed
쐃 • Collect the sample by centrifugation at 2,000 × g for 5 min in a
standard table top centrifuge. Poor elution Water has the If you use your own water or buffer
• Resuspend the pellet in 200 l 10 mM Tris, pH 8.0. of nucleic wrong pH to elute nucleic acids from Filter
acids with Tube, be sure it has the same pH as
쐇 Add 4 l Lysozyme (50 mg/ml), incubate for 10 min at 37°C. water the Elution Buffer supplied in the
쐋 Add 400 l Lysis/-Binding Buffer (green cap) and mix well. kit.
쐏 Combine the High Pure Filter Tube and the Collection Tube and Absorbance Glass fibers, which 1. Remove High Pure Filter Tube
pipette the sample in the upper reservoir. (A 260 nm ) might coelute with from tube containing eluted
reading of nucleic acid, scatter sample and spin sample for 1
쐄 Centrifuge for 15 s at 8,000 × g in a standard table top centri- product too light min at maximum speed.
fuge, discard the flowthrough and again combine the Filter Tube high 2. Transfer supernatant into a new
and the used Collection Tube. tube without disturbing the glass
쐂 • Pipette 90 l DNase Incubation Buffer (white cap) into a sterile fibers at the bottom of the origi-
reaction tube, add 10 l DNase I, mix and pipette the solution nal tube.
in the upper reservoir of the Filter Tube. Low RNA High levels of • Be careful to create an RNase-
• Incubate for 60 min at +15 to +25°C. yield RNase activity free working environment.
쐆 Follow protocol 2.2 as described for cultured cells from step 7. • Process starting material immedi-
ately or store it at –80°C until it
can be processed.
3. Results • Use eluted RNA directly in down-
stream procedures or store it
Source Average yield (µg) immediately at –80°C.
Cultured cells (106 cells)
K562 15 g 5. Additional Information on this Product
Human blood (200 – 500 l) Not measurable, use in RT-PCR only 5.1 How this Product Works
Yeast (108 cells) Isolation of RNA is a prerequisite for the analysis of gene expression.
S. cerevisiae 20 g Frequently applied techniques like reverse transcriptase-PCR (RT-
9 PCR), northern blotting, RNase protection and primer extension
Bacteria (10 cells) require the use of intact, undegraded RNA from different sample
E. coli 50 g materials like cultured cells, blood, yeast and bacteria.
B. subtilis 35 g
Test principle
Isolated total RNA can be used directly in first-strand cDNA synthesis.
햲 Cultured cells are lysed by a special Lysis/-Binding buffer. At the
Depending on the expression level of the target mRNA to be analysed,
same time, RNases are inactivated.
it is recommended, to use 1 – 10 l in the RT reaction. Perform an RT-
minus control reaction (by omitting addition of reverse transcriptase L Other sample materials require a specific pre-lysis treatment.
to the cDNA synthesis reaction). This will indicate whether your RNA 햳 Nucleic acids are bound to the glass fibers pre-packed in the
sample contain residual genomic DNA by causing false-positive High Pure Filter Tube.
results.
햴 Residual contaminating DNA is digested by DNase I, applied
directly on the glass fiber fleece.
햵 Bound nucleic acids are washed with a special Inhibitor Removal
Buffer to get rid of RT-PCR inhibitory contaminants.
햶 Washing of bound nucleic acids, purification from salts, proteins
and other cellular impurities.
햷 RNA is recovered using the Elution Buffer.

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References 6.3 Ordering Information
1 Vogelstein, B., & Gillespie, D. (1979) Preprative an analytical Roche Applied Science offers a large selection of reagents and systems for life
science research. For a complete overview of related products and manuals,
purification of DNA from agarose Proc. Natl. Acad. Sci. USA 76, please visit and bookmark our home page www.roche-applied-science.com
615-619. and our Special Interest Sites including:
2 Geiß, U. et al. (2003) Detection of Prochlorothrix in Brackish • Nucleic Acid Isolation and Purification:
Waters by Specific Amplification of pcb Genes Applied and Envi- http://www.roche-applied-science.com/napure
ronmental Microbiology 69(10), 6243-6249. • PCR - Innovative Tools for Amplification:
http://www.roche-applied-science.com/pcr
3 Schettler, V. et al (2003) No acute impact of haemodialysis treat- • http://www.roche-applied-science.com/sis/rtpcr
ment on free radical scavenging enzyme gene expression in
white blood cells Journal of Internal Medicine 253(2), 201-207. Product Pack Size Cat. No.
4 Bouzin , C. et al. (2003) The Onecut Transcription Factor Hepato- Associated High Pure 16 System Viral 96 reactions 12 011 816 001
cyte Nuclear Factor-6 Controls B Lymphopoiesis in Fetal Liver Kits Nucleic Acid Kit
The Journal of Immunology 171, 1297-1303. High Pure Viral Nucleic 100 reactions 12 011 875 001
5 Lillicarp, MS. et al. (2004) T cell recognition of a highly Acid Buffer Set
conserved epitope in heat shock protein 60: self-tolerance High Pure PCR Template 100 11 796 828 001
maintained by TCR distinguishing between asparagine and Preparation Kit purifications
aspartic acid International Immunology, 16(3), 405-414. LightCycler® RNA Master 1 kit 03 018 954 001
6 Boyman, O. et al. (2004) Spontaneous Development of Psoriasis HybProbe (96 reactions)
in a New Animal Model Shows an Essential Role for Resident T LightCycler® RNA Master 1 kit 03 064 760 001
Cells and Tumor Necrosis Factor-α J. Exp. Med. 199 (5), 731- SYBR Green I (96 reactions)
736. LightCycler® RNA Amplifi-1 kit 12 015 145 001
cation Kit HybProbe (96 reactions)
Quality Control LightCycler® RNA Amplifi- 1 kit 12 015 137 001
1 × 106 K 562 cells are treated as described in the protocol for cultured cation Kit SYBR Green I (96 reactions)
cells. RNA yield is determined by measuring the optical density at 260 LightCycler® Control 1 kit 12 158 841 001
nm. At least 10 g of total RNA are isolated. Integrity and size distribu- Kit RNA (50 reactions)
tion are examined by the banding pattern of ribosomal RNA in a dena- High Pure PCR Product 50 purifications 11 732 668 001
turing agarose gel. 100 ng of isolated total RNA is used in first strand Purification Kit 250 purifications 11 732 676 001
synthesis with reverse transcriptase M-MuLV* and p(dT)15* as a
Single Reverse Transcriptase, 500 U 11 062 603 001
primer. In the following PCR, accomplished with Expand High Fidelity reagents M-MuLV
PCR System* and specific primers for glyceraldehyde 3-phosphate
dehydrogenase (G3PDH), the expected amplification product of 983 Reverse Transcriptase 500 U 11 495 062 001
bp is obtained. AMV 1 000 U 10 109 118 001
Absence of contaminating DNA is examined by a PCR without preced- Transcriptor Reverse 250 U 03 531 317 001
ing RT-reaction; no amplification product is obtained. All kit compo- Transcriptase 500 U 03 531 295 001
2 000 U 03 531 287 001
nents are function tested for absence of RNases.
Red Blood Cell Lysis 100 ml 11 814 389 001
Buffer
6. Supplementary Information Buffers in a Box, 4l 11 666 789 001
Premixed PBS Buffer, 10×
6.1 Conventions
Lysozyme 10 g 10 837 059 001
Text Conventions
To make information consistent and memorable, the following text conventions 6.4 Trademarks
are used in this Instruction Manual: HIGH PURE, LIGHTCYCLER, HYBPROBE and EXPAND are trademarks of
Roche.
Text Convention Use Triton is a trademark of Rohm and Haas Company, Philadelphia, PA, USA.
SYBR is a trademark of Molecular Probes Inc., Eugene, OR, USA.
Numbered stages Stages in a process that usually occur in Other brands or product names are trademarks of their respective holders.
labeled c, w, etc. the order listed
Numbered instructions Steps in a procedure that must be per-
labeled w, o, etc. formed in the order listed
Asterisk * Denotes a product available from Roche
Applied Science
Symbols
In this Instruction Manual symbols are used as an optical signal to point out
important information:
Symbol Description
Information Note:
L Additional information about the current topic or proce- Contact and Support
dure.
To ask questions, solve problems, suggest enhancements or report new
Important Note: applications, please visit our Online Technical Support Site at:
N Information critical to the success of the procedure or use
www.roche-applied-science.com/support
of the product.
To call, write, fax, or email us, visit the Roche Applied Science home page,
6.2 Changes to Previous Versions www.roche-applied-science.com, and select your home country. Country-
• Editorial Changes specific contact information will be displayed. Use the Product Search func-
tion to find Pack Inserts and Material Safety Data Sheets.

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Germany