Total RNA Extraction With TRIZOL Reagent 10172007

Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy
Mini Kit
DGC, Indiana University- October 11, 2007
Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN
RNeasy Mini Kit
Includes protocol for RNA Quantitative and Qualitative Assessment
Jacqueline Ann Lopez and Elizabeth Bohuski
Based on DGRC protocol by Kevin Bogart, Jason Conaty, and Justen Andrews
INTRODUCTION
The utilization of microarrays for transcription analysis, cDNA synthesis, Q-RT PCR, and Northern
Analysis requires firstly the extraction of RNA from a biological source of interest. We describe this
procedure in detail along with RNA quality control measures. As per all steps involved in RNA isolation, it
is essential to avoid latex gloves use only nitrile gloves. It is likewise important to guard against sources of
dust and nucleases. We recommend using barrier pipette tips, and filtering all solutions (except those that are
purchased as sterile and nuclease-free). Wipe down pipetters and bench with RNase Zap as instructed by
manufacturer.
MATERIALS
Item Description
Company
Cat. No.
Trizol Reagent
Chloroform
Rneasy Mini Kit (50)
Rnase-free Dnase set (50)
Rnase Zap
UltraPure Dnase/Rnase-Free Distilled water
Ethanol (100%) *
1.5 mL micro-centrifuge tubes *
Disposable pestles; 1.5mL, Plastic *
Aerosol Resistant Tips (1000E) *
Aerosol Resistant Tips (200) *
Eppendorf Microcentrifuge 5415 R
Invitrogen
EMD Chemicals
Qiagen
Qiagen
Ambion
Invitrogen
AAPER Alcohol
VWR
VWR
Molecular BioProducts
Eppendorf
15596-025
CX1055-6
74104
79254
AM9780/AM9782
10977-015
* Dnase / Rnase-free grade
101093-206
KT749521-1590
REF2079E
REF2069
REF2149P
REF2139
-------
Unit Size
100 mL
500 mL
50 rxn
50 rxn
250 mL
500 mL
500 mL
500 tubes
100 pestles
100 Tips
96 Tips
96 Tips
96 Tips
-------
Mini Kit
PROCEDURE
Preparations
1. Cool Centrifuge to 4 oC.

Cooled centrifuge is necessary for RNA isolation.
2. Decontaminate work space and equipment with RNase ZAP following manufacturers
instructions (printed on the bottle).
3. Setup the following for each sample: one RNeasy mini-spin column, one 2-mL collection
tube, and two 1.5mL micro-centrifuge tubes (one supplied in RNeasy Mini Kit and one
supplied by user).
Label a single mini-spin column with collection tube attached, a second 2-mL collection tube,
and a single micro-centrifuge tube per sample. These materials are provided in the kit and
should be handled with Rnase-free gloves to prevent contamination. The user will supply a
second 1.5mL micro-centrifuge tube (Rnase/Dnase-Free, grade).
4. Prepare Buffer RPE. (Included in RNeasy Kit)

RPE buffer is supplied as a concentrate. Before using for the first time, add 4 volumes of 96% 100% ethanol, as indicated on the bottle, to obtain the working solution. Solution can be stored
at room temperature. Be sure to check the YES box on the cap to indicate that ethanol was
added. Write the date on the bottle, as well.
5. On ice, prepare DNase stock solution from the QIAGEN Rnase-free Dnase set.
IMPORTANT: DNase enzyme is supplied as a lyophilized protein. Dnase enzyme is sensitive to
physical denaturation. Do not vortex
Before using for the first time, add 550l of nuclease-free water (provided in the Rnase-free
Dnase set). Mix by inverting gently.
To avoid repetitive freezing and thawing of the enzyme, make aliquots of a desired volume.
Store aliquots for up to 6 months at -20 oC.
o For example, I process 8-extraction samples at a time and each sample requires 5 l of
the enzyme. So I make aliquots of 40 l in 0.5mL Rnase-free micro-centrifuge tubes.
o (i.e. 5 l x 8-extraction samples = 40 l)
Do not freeze aliquot after thawing. DNase enzyme is sensitive to physical denaturation.
Thawed enzyme may be store for up to 1 week at 4 oC.
Store Buffer RDD (provided in the Rnase-free Dnase set) at 4 oC.
Recommend that Buffer RDD is added to the Dnase enzyme just prior to use.
Mini Kit
Cell Lyses and RNA Extraction with Organics

FOR LIVE OR FROZEN DAPHNIA TISSUE
1. Determine the amount of tissue.
For FROZEN samples, weighing the sample using a metric balance or scale is the most accurate
method.
For LIVE samples, the number of individuals is a sufficient to estimate the volume of
tissue. Assume that a single mature female equals 1 l in volume.
2. Add one volume of TRIZOL Reagent. The sample volume should not exceed 10% of the
volume of TRIZOL Reagent used for homogenization.
For 10 mg 100 mg or 10 l 100 l of Daphnia, add 500 l of TRIZOL Reagent.
For 1 mg 10 mg or 1 l 10 l of Daphnia, add 400 l of TRIZOL Reagent.
3. Using a blue plastic pestle, homogenize tissue sample in the TRIZOL Reagent.
Disruption and homogenization of tissue can be performed by crushing Daphnia with disposable
plastic pestle by hand (~5 minutes) or using a battery operated homogenizer.
Incomplete homogenization will lead to significantly reduced yields and can clog RNeasy column.
4. Wash down the blue plastic pestle with a second volume of TRIZOL Reagent (equivalent
to amount used for step 2 above).
5. Mix by inverting 10 times.
NOTE: Tissue homogenized in 0.8 1 mL of TRIZOL Reagent can be stored at -80 oC for up to
1 month.
6. Incubate homogenized tissue in TRIZOL Reagent for 5 minutes at room temperature
(15C to 30C) to permit the complete dissociation of nucleoprotein complexes.
7. Add 200 l of Chloroform per 1 mL of TRIZOL Reagent. Shake tube vigorously by hand
for 15 seconds and incubate for 2 to 3 minutes at 15C to 30C.
8. Centrifuge at 11,600 rcf for 15 minutes at 4C
Centrifuge the samples at no more than 12,000 g for 15 minutes at 2 to 8C. Following
centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a
colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the
aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization.
9. Transfer upper aqueous phase (approx. 540 l) to a NEW Rnase-free 1.5 mL microcentrifuge tube (supplied by user). Save the organic phase if isolation of DNA or protein
Mini Kit
is desired (See manufactures instructions for DNA or protein isolation).
10. Precipitate RNA by adding 0.5x volume of 100% room temperature Ethanol.
11. Mix by gently inverting 10 times.
RNA Purification & On-column DNase treatment with RNeasy Mini Kit
Note: The following steps are modified from the protocol outlined in detail in "RNeasy Mini
Handbook; RNeasy Mini Protocol for RNA Clean-up," p. 79-81,
(http://www.qiagen.com/literature/rnalit.asp#mini).
1. Transfer precipitated RNA to a Qiagen RNeasy mini-spin column.

Max. loading volume: 700 l
Max. binding capacity: 100 g
Exceeding the binding capacity of the mini-spin column (> 100 g) will significantly reduce
yield and quality of the recovered RNA.
For samples with expected yields > 100 g of Total RNA, divide the precipitate between two
RNeasy mini-spin columns.
2. Centrifuge at 9,600 rcf for 30sec at room temperature (15 30 C). Discard flow through.
If the volume of precipitated RNA exceeds 700l, load aliquots successively onto the RNeasy
mini-spin column and repeat centrifugation. Discard flow through after each centrifugation
step. Replace mini-spin column into same 2 mL collection tube.
3. On-Column Dnase Treatment:

If on-column DNase treatment using RNase-Free DNase Set is NOT desired, Ambion Dnafree (AM1906, 50rxn) is a recommended alternative for removal of contaminating DNA
from RNA preparations. To continue RNA purification, increase the amount of Buffer RW1
to 700 l. Centrifuge at 9,600 rcf for 30sec at 4C. Discard the flow through. Proceed to
Step 4 of this section.
Wash column with 350 l of Buffer RW1.
Centrifuge at 9,600 rcf for 30sec at room temperature (15 30 C). Discard flow
through. Replace column into same 2 mL collection tube.
Add 35 l Buffer RDD to 5 l DNase I stock solution. Mix by gently pipetting.
Pipet the DNase I incubation mix (40 l) directly onto the RNeasy silica-gel membrane
of the RNeasy mini-spin column.
Incubate for 15 minutes room temperature (15 30 C).
Add to column with 350 l of Buffer RW1.
Centrifuge at 9,600 rcf for 30sec at room temperature (15 30 C). Discard flow
through.
Mini Kit
4. Wash column with 500 l of Buffer RPE.
Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use. See
preparation section at the beginning of this protocol for details.
Following the centrifugation of Buffer RPE, remove the RNeasy silica-gel mini column from the
collection tube carefully so the column does not contact the flow-through as this will result in
carryover of ethanol.
5. Centrifuge at 9,600 rcf for 30sec at room temperature (15 30 C). Carefully remove
mini-spin column. Discard flow through. Replace column into same 2 mL collection tube.
6. Wash column with 500 l of Buffer RPE.
7. Centrifuge at 9,600 rcf for 30sec at room temperature (15 30 C). Carefully remove
mini-spin column. Discard flow through.
8. Place column in NEW 2 mL collection tube (supplied in RNeasy Mini Kit).
9. Centrifuge at 16,000 rcf for 2minutes at room temperature (15 30 C). Carefully remove
mini-spin column.
It is important to remove trace amount Buffer RPE. Otherwise this will result in ethanol carry over
and reduce the quality of the eluted sample.
10. Transfer RNeasy column to a NEW 1.5 mL micro-centrifuge tube (supplied in RNeasy
Mini Kit).
11. To elute, pipet 30 l RNase-Free water directly onto silica-gel membrane. Incubate for 1
minute at room temperature.
12. Centrifuge at 9,600 rcf for 1 minute at room temperature (15 30 C).
If the expected RNA yield is >30 g, repeat the elute step (Step 9 & 10) as described with a second
volume of RNase-free water. Collect and measure each elution separately on Nanodrop. Second
elution is sometimes less pure.
13. Store RNA at -80C.

RNA samples eluted with water can be stored for up to 1 month at -80C.
For long term storage, RNA can be precipitated and stored in ethanol for up to 2 years.
o Ethanol Precipitation
0.1x volume 5 M NH4OAc
2.5x volume 100 % Ethanol
Mix by inverting 10 times.
Store at -80C.
Mini Kit
RNA Quantitative Assessment: Determine concentration

Item Description
Company
Cat. No.

Kimwipes (small)
Total RNA (eluted in water)
Invitrogen
10977-015
REF2139
Unit Size
500 mL
96 Tips
1. Launch Nanodrop software to start application and adjust setting for RNA quantification.
Select Nucleic Acid.
2. A prompt will appear.

3. Buff top and bottom pedestal with a Kimwipe. Apply pressure gently and stroking 10
times with Kimwipe.
4. Load 1.3 l of nuclease-free water to pedestal. Lower arm and click OK.
5. Select Sample Type from drop-down menu.
Sample type: RNA 40
Extinction coefficient = 40
6. Buff pedestal both top and bottom of pedestal after every measurement.
7. To BLANK, apply to the pedestal 1.3 l of elution buffer in which RNA is dissolved.
If water was used to elute the sample, the blank measurement is done with the same water sample
used to elute RNA during purification. If another elution buffer was used, that is the buffer used to
calibrate the instrument.
8. Click BLANK to calibrate the instrument.

9. Instrument is now ready to measure concentration of Total RNA sample. Be sure to enter
a Sample Name for each sample and measurement reading.
10. To make sure instrument is calibrated properly, apply 1.3 l of Elution buffer and click
Measure. Reading should be close to zero.
11. To quantify sample, apply 1.3 l of Total RNA sample to pedestal and click Measure.
The concentration of RNA is determined by measuring the absorbance at 260 nm (A260). To ensure
significance, the reading at 260 (A260) should be greater than 0.15.
Pure RNA: 260/280 ratio of ~ 2.0
If the ratio is lower, this may indicate the presence of protein, phenol or other contaminants that
absorb at or near 280 nm.
Pure RNA: 260/230 ratio range of 1.8 2.2
If the ratio is low, this may indicate the presence of co-purified contaminants.
Mini Kit
Pure RNA: Smooth curve.
Trouble shooting:
Make sure sample pedestal is clean. Use 2.0ul of deionized water to wash pedestal and wipe dry
with a kimwipe.
Redo the blank setup. If blank measurement is not done properly, strange results will occur.
RNA sample may not be homogenized. Gently mixing Total RNA sample by finger-flicking the
micro-centrifuge tube prior to measuring the concentration is recommended. Re-measure the
sample.
Use a 1.5 2.0 ul sample size when measuring. Strange results occur when the liquid sample
column is not completely formed during the measurement. While making a measurement,
visually confirm the water column is completely formed.
If this does not resolve the issue, RNA sample may need to be re-purified. Recommend
QIAGEN RNA Mini Kit: RNA Cleanup protocol (See Manufacturers Manual).
Mini Kit
Total RNA Qualitative Assessment: Bio Analyzer

It is likewise important to guard against sources of dust and nucleases. We recommend using barrier pipette
tips, and filtering all solutions (except those that are purchased as sterile and nuclease-free). Wipe down all
electrophoresis equipment, pipettors and bench with RNase Zap as instructed by manufacturer.
Item Description
Company
Cat. No.
Rnase Zap
PCR strip w/ cap (8 tubes/strip) *
Eppendorf Microcentrifuge 5424
NanoChip Agilent RNA 6000 Nano Kit
0.5 mL Safe Lock micro-centrifuge tubes *
Ambion
Invitrogen
VWR
Eppendorf
Aligent
Agilent
AM9780/AM9782
10977-015
20170-004
REF2079E
REF2069
REF2149P
REF2139
022620401
5067-1511
kit
Unit Size
250 mL
500 mL
125 strips
100 Tips
96 Tips
96 Tips
96 Tips
------25 chips
1. Prepare Total RNA sample (vol = 1.5 l) to a final concentration of 300 ng/l.
Concentrate 400 ng Total RNA to a volume of 1.5 l in a 0.2 mL micro-centrifuge tube. Mix by
gently pipetting. Centrifuge briefly to collect the contents
Keep sample on ice.
2. Set up Bioanalyzer software and adjust settings for Total RNA quantification according to
manufactures instructions.
Follow manufacturers instructions to assess the quality of the Total RNA.
Dilute the sample to a concentration of 300 ng/ l. Over loading the chip with Total RNA leads to
inaccurate assay results.
3. Interpretation of Bioanalyzer results:

Assess quality of Total RNA from electrophrograms of RNA ribosomal peaks.
High Quality RNA Profile
Distinct 18S and 28S peaks.
Low noise between peaks
Minimal low-molecular weight

contamination.
Minimal high-molecular weight

contamination. High molecular weight contamination may indicate the presence
of a contaminant (i.e. DNA).
Mini Kit
Moderate to High Quality RNA Profile
Distinct 18S and 28S peaks, but peaks are even in height.
Low noise between peaks
Minimal low-molecular weight

contamination.

contamination. High molecular weight
contamination may indicate the presence of a
contaminant (i.e. DNA).
Moderate to Low Quality RNA Profile
28S peak is considerably lower than 18S peak.
Low-molecular weight contamination may

indicate degradation.

contamination may indicate the presence of
a contaminant (i.e. DNA).
Low Quality RNA Profile
28S peak is not visible compared to 18S peak.
Abundant low-molecular weight

contamination may indicate degradation.

contamination may indicate the presence of
a contaminant (i.e. DNA).
Mini Kit
Freezing Daphnia: Whole Body Tissue Preparation

MATERIALS
Item Description
Company
Cat. No.

Pasteur pipet
Liquid Nitrogen
Long Forceps
VWR
----------------
101093-206
-------------------
Unit Size
500 tubes
-------------------
1. Collect Daphnia sample in 1.5 mL micro-centrifuge tube.

When collecting from a large culture (gallon jars), slowly pour culture into a smaller glass dish or
beaker.
When collecting from small cultures (beakers), collect adults directly from the culture.
2. Using a sterile Pasteur pipette, collect desired sample in a labeled 1.5 mL micro-centrifuge
tube.
Daphnia can be temporarily stored in this collection tube for up to 5 minutes while continuing with
the remainder of the samples collections. Be sure to leave approx.1 mL of water in the tube.
3. Remove water with Pasteur pipette. Freeze Daphnia in liquid nitrogen and store at -80 oC
until proceeding to Total RNA isolation and purification.
Gently remove water from 1.5 mL micro-centrifuge tube with Pasteur pipette. Place the tip of a
sterile pipette at the bottom of the tube. Expel air gently to displace any animals obstructing the
pipette tip. Once all water is removed from sample, immediately freeze tube of tissue in liquid
nitrogen. Remove tube of frozen tissue from liquid nitrogen with forceps. Store at -80 oC.
Mini Kit
Total RNA Qualitative Assessment: Protocol for RNA Gel with Guanidine-HCl
It is likewise important to guard against sources of dust and nucleases. We recommend using barrier pipette
tips, and filtering all solutions (except those that are purchased as sterile and nuclease-free). Wipe down all
electrophoresis equipment, pipettors and bench with RNase Zap as instructed by manufacturer.
Unit Size
Item Description
Company
Cat. No.
Rnase Zap
PCR strip w/ cap (8 tubes/strip) *
Eppendorf Microcentrifuge 5415 R
Guanidine hydrochloride (powder)
Ethdium Bromide (powder)
Formamide
1X TE buffer (pH 7.0) *
1 X TBE buffer *
Agarose (powder)
6 X Loading Dye *
Electrophoresis apparatus
Casting tray and comb
Ambion
Invitrogen
VWR
VWR
Eppendorf
Sigma-Aldrich
Sigma-Aldrich
AppliChem GmbH
-------------------------------
AM9780/AM9782
10977-015
101093-206
20170-004
REF2079E
REF2069
REF2149P
REF2139
------G3272
E7637-1G
A0937,0500
-------------------------------------
250 mL
500 mL
500 tubes
125 strips
100 Tips
96 Tips
96 Tips
96 Tips
------100 grams
100 grams
500 mL
-------------------------------------
1. Prepare a 1.5% w/v Agarose gel with 1X TBE with 20 mM Guanidine-HCl.

Add 0.75g Agarose to 50 mL of 1X TBE buffer for a 50 mL volume 1.5% w/v gel.
Boil to dissolve Agarose.
Adjust volume with water and allow solution to cool to 55C.
2. Add Guanidine-HCl to a final concentration of 20mM.

For a 50 mL volume 1.5% w/v gel, add 1 mL of 1M guanidine-HCl.
3. Add 2 l Ethidium Bromide (10mg/mL) to cool Agarose solution.

Ethidium Bromide is a carcinogen and should be handled according to safety protocols.
4. Pour gel and let solidify.
Pour gel solution into casting tray. Push any air bubbles using a sterile tooth pick to be bottom of the
gel. Air bubbles will interfere with the nucleic acid separate and lead to poor results.
5. Prepare 1 g Total RNA sample for electrophoresis.

Concentrate 1 g of RNA to a volume of 1 l.
6. To 1 l concentrated Total RNA sample,
add 4 l Formamide-TE buffer

Denature by heating at 70C for 2 minutes
Briefly centrifuge to collect sample.
Add 1 l of Loading dye.
Mini Kit
7. Prepare gel for electrophoresis.
Remove comb, and submerge gel into electrophoresis chamber filled with electrophoresis buffer.
8. Load one sample per gel into the wells.

9. Electrophoresis for 1.0 hours at 97 mV.
10. Visualize RNA ribosomal bands with UV shadowing.
11. The high molecular weight band should be about twice as intense as the low molecular
weight band. Little smearing between bands.
12. Low-molecular weight contamination may indicate RNA degradation. High-molecular
weight contamination may indicate DNA contamination.
Mini Kit
Solutions
Formamide-TE buffer
Add 300 l formamide to 100 l 1x TE
1M Guanidine-HCL
Dissolve 4.77 g Guanidine-HCL in 50 mL of filtered, distilled deionized water.
10 mg/mL Ethidium bromide recipe
Dissolve 1.0 g Ethidium bromide in 100 mL filtered, distilled deionized water
When dissolved, wrap container in aluminum foil and keep in the dark. Use a dark glass bottle, if
possible.
6 X Gel loading buffer with glycerol recipe
25.0 mg Bromophenol blue
25.0 mg Xylene cyanol FF
3.0 mL glycerol.
Bring to a volume of 10 mL with distilled water.
Store at 4C.
1x TBE electrophoresis buffer recipe (working solution)
To 700 mL of filtered, distilled deionized water, add
54.0 g Tris base
27.5 g Boric acid
20.0 mL 0.5 M EDTA (pH 8.0)
Bring to a final volume of 1 L.
Autoclave to sterilize.
10 X TBE electrophoresis buffer recipe (Stock Solution)
To 700 mL of filtered, distilled deionized water, add
1.0 g NaOH
108.0 g Tris Base
55.0 g Boric Acid
7.4 g EDTA (disodium salt)
Stir to dissolve.
Bring up final volume of 1 L.
Autoclave to sterilize.
1X TE
To 990 mL deionized, distilled nuclease free water,
Add 10mL 1M Tris-HCl (pH 8.0)
Add 200l 0.5 M EDTA
5 M EDTA (pH 8.0): 500 mL
Dissolve 95.05 g of Na2EDTA in 400 mL deionized, distilled nuclease free water.
Adjust pH to 8.0 with 6 N NaOH.
Then bring volume to 500 mL and sterilize by autoclaving.
20 minute sterilization, 20 minute exhauste.
1M Tris-HCl, pH 8.0
12.1g Tris base (MW 121.14) in 100mL H2O (adjust pH with concentrated HCl)
Mini Kit

Total RNA Extraction With TRIZOL Reagent 10172007

Transféré par

Informations du document

Copyright

Formats disponibles

Partager ce document

Partager ou intégrer le document

Options de partage

Avez-vous trouvé ce document utile ?

Ce contenu est-il inapproprié ?

Droits d'auteur :

Formats disponibles

Total RNA Extraction With TRIZOL Reagent 10172007

Transféré par

Droits d'auteur :

Formats disponibles

Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasy

* Dnase / Rnase-free grade

1. Cool Centrifuge to 4 oC.

4. Prepare Buffer RPE. (Included in RNeasy Kit)

Cell Lyses and RNA Extraction with Organics

1. Transfer precipitated RNA to a Qiagen RNeasy mini-spin column.

3. On-Column Dnase Treatment:

13. Store RNA at -80C.

RNA Quantitative Assessment: Determine concentration

UltraPure Dnase/Rnase-Free Distilled water

* Dnase / Rnase-free grade

2. A prompt will appear.

8. Click BLANK to calibrate the instrument.

Total RNA Qualitative Assessment: Bio Analyzer

3. Interpretation of Bioanalyzer results:

High Quality RNA Profile

Distinct 18S and 28S peaks.

Low noise between peaks

Minimal low-molecular weight

Minimal high-molecular weight

Moderate to High Quality RNA Profile

Low noise between peaks

Minimal low-molecular weight

Minimal high-molecular weight

Moderate to Low Quality RNA Profile

28S peak is considerably lower than 18S peak.

Low-molecular weight contamination may

Minimal high-molecular weight

Low Quality RNA Profile

28S peak is not visible compared to 18S peak.

Abundant low-molecular weight

Minimal high-molecular weight

Freezing Daphnia: Whole Body Tissue Preparation

1.5 mL micro-centrifuge tubes *

* Dnase / Rnase-free grade

1. Collect Daphnia sample in 1.5 mL micro-centrifuge tube.

1. Prepare a 1.5% w/v Agarose gel with 1X TBE with 20 mM Guanidine-HCl.

2. Add Guanidine-HCl to a final concentration of 20mM.

3. Add 2 l Ethidium Bromide (10mg/mL) to cool Agarose solution.

5. Prepare 1 g Total RNA sample for electrophoresis.

6. To 1 l concentrated Total RNA sample,

add 4 l Formamide-TE buffer

8. Load one sample per gel into the wells.

weight contamination may indicate DNA contamination.

Vous aimerez peut-être aussi