Total RNA Extraction With TRIZOL Reagent 10172007

8/10/2019 Total RNA Extraction With TRIZOL Reagent 10172007

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Protocol: Total RNA Extraction with TRIZOL Reagent and Purification with QIAGEN RNeasyMini KitDGC, Indiana University- October 11, 2007

Total RNA Extraction with TRIZOL Reagent and Purification with QIAGENRNeasy 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

INTRODUCTIONThe utilization of microarrays for transcription analysis, cDNA synthesis, Q-RT PCR, and NorthernAnalysis 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, itis essential to avoid latex gloves use only nitrile gloves.It is likewise important to guard against sources ofdust and nucleases. We recommend using barrier pipette tips, and filtering all solutions (except those that arepurchased as sterile and nuclease-free). Wipe down pipetters and bench with RNase Zap as instructed bymanufacturer.

MATERIALSItem Description Company Cat. No. Unit Size

Trizol Reagent Invitrogen 15596-025 100 mL

Chloroform EMD Chemicals CX1055-6 500 mL

Rneasy Mini Kit (50) Qiagen 74104 50 rxn

Rnase-free Dnase set (50) Qiagen 79254 50 rxn

Rnase Zap Ambion AM9780/AM9782 250 mL

UltraPure Dnase/Rnase-Free Distilled water Invitrogen 10977-015 500 mLEthanol (100%) * AAPER Alcohol 500 mL

1.5 mL micro-centrifuge tubes * VWR 101093-206 500 tubes

Disposable pestles; 1.5mL, Plastic * VWR KT749521-1590 100 pestles

Aerosol Resistant Tips (1000E) * Molecular BioProducts REF2079E 100 Tips

Aerosol Resistant Tips (200) * Molecular BioProducts REF2069 96 TipsAerosol Resistant Tips (20) * Molecular BioProducts REF2149P 96 TipsAerosol Resistant Tips (10) * Molecular BioProducts REF2139 96 TipsEppendorfMicrocentrifuge 5415 R Eppendorf ------- -------

* Dnase / Rnase-free grade


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PROCEDURE

Preparations1. 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 andshould be handled with Rnase-free gloves to prevent contamination. The user will supply asecond 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 storedat room temperature. Be sure to check the YES box on the cap to indicate that ethanol wasadded. 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 tophysical 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 -20oC.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 4oC. Recommend that Buffer RDD is added to the Dnase enzyme just prior to use.


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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 1l 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 ofDaphnia, add 500 l of TRIZOL Reagent.

For 1 mg 10 mg or 1 l 10 l ofDaphnia, 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 crushingDaphniawith 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 -80oC 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 gfor 15 minutes at 2 to 8C. Followingcentrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and acolorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of theaqueous 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 micro-

centrifuge tube (supplied by user). Save the organic phase if isolation of DNA or protein


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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 KitNote: The following steps are modified from the protocol outlined in detail in "RNeasy MiniHandbook; 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 reduceyield and quality of the recovered RNA.

For samples with expected yields > 100 g of Total RNA, divide the precipitate between twoRNeasy 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 RNeasymini-spin column and repeat centrifugation. Discard flow through after each centrifugationstep. 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 Dna-free (AM1906, 50rxn) is a recommended alternative for removal of contaminating DNAfrom RNA preparations. To continue RNA purification, increase the amount of Buffer RW1to 700 l. Centrifuge at 9,600 rcf for 30sec at 4C.Discard the flow through. Proceed toStep 4of 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.


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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 incarryover 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 overand 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 secondvolume of RNase-free water. Collect and measure each elution separately on Nanodrop. Secondelution 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.


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RNA Quantitative Assessment: Determine concentration

Item Description Company Cat. No. Unit Size

UltraPure Dnase/Rnase-Free Distilled water Invitrogen 10977-015 500 mL

Aerosol Resistant Tips (10) * Molecular BioProducts REF2139 96 TipsKimwipes (small)

Total RNA (eluted in water)


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 ensuresignificance, 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 thatabsorb 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.


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Pure RNA: Smooth curve. Trouble shooting:

Make sure sample pedestal is clean. Use 2.0ul of deionized water to wash pedestal and wipe drywith 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 thesample.

Use a 1.5 2.0 ul sample size when measuring. Strange results occur when the liquid samplecolumn 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. RecommendQIAGEN RNA Mini Kit: RNA Cleanup protocol (See Manufacturers Manual).


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Total RNA Qualitative Assessment: Bio Analyzer

It is likewise important to guard against sources of dust and nucleases. We recommend using barrier pipettetips, and filtering all solutions (except those that are purchased as sterile and nuclease-free). Wipe down allelectrophoresis equipment, pipettors and bench with RNase Zap as instructed by manufacturer.




PCR strip w/ cap (8 tubes/strip) * VWR 20170-004 125 strips


Aerosol Resistant Tips (200) * Molecular BioProducts REF2069 96 TipsAerosol Resistant Tips (20) * Molecular BioProducts REF2149P 96 TipsAerosol Resistant Tips (10) * Molecular BioProducts REF2139 96 TipsEppendorfMicrocentrifuge 5424 Eppendorf 022620401 -------

NanoChip Agilent RNA 6000 Nano Kit Aligent 5067-1511 25 chips

0.5 mL Safe Lock micro-centrifuge tubes * Agilent kit

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 bygently 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 weightcontamination.

Minimal high-molecular weight

contamination. High molecular weight contamination may indicate the presence

of a contaminant (i.e. DNA).


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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).


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Freezing Daphnia: Whole Body Tissue Preparation

MATERIALSItem Description Company Cat. No. Unit Size


Pasteur pipet ------ ------- -------

Liquid Nitrogen ------ ------- -------

Long Forceps ------ ------- -------


1. CollectDaphniasample in 1.5 mL micro-centrifuge tube.

When collecting from a large culture (gallon jars), slowly pour culture into a smaller glass dish orbeaker.

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.

Daphniacan be temporarily stored in this collection tube for up to 5 minutes while continuing withthe remainder of the samples collections. Be sure to leave approx.1 mL of water in the tube.

3. Remove water with Pasteur pipette. FreezeDaphniain liquid nitrogen and store at -80oC

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 asterile pipette at the bottom of the tube. Expel air gently to displace any animals obstructing thepipette tip. Once all water is removed from sample, immediately freeze tube of tissue in liquidnitrogen. Remove tube of frozen tissue from liquid nitrogen with forceps. Store at -80oC.


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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 pipettetips, and filtering all solutions (except those that are purchased as sterile and nuclease-free). Wipe down allelectrophoresis equipment, pipettors and bench with RNase Zap as instructed by manufacturer.





PCR strip w/ cap (8 tubes/strip) * VWR 20170-004 125 strips


Aerosol Resistant Tips (200) * Molecular BioProducts REF2069 96 TipsAerosol Resistant Tips (20) * Molecular BioProducts REF2149P 96 TipsAerosol Resistant Tips (10) * Molecular BioProducts REF2139 96 Tips

EppendorfMicrocentrifuge 5415 R Eppendorf ------- -------Guanidine hydrochloride (powder) Sigma-Aldrich G3272 100 grams

Ethdium Bromide (powder) Sigma-Aldrich E7637-1G 100 grams

Formamide AppliChem GmbH A0937,0500 500 mL

1X TE buffer (pH 7.0) * ------ ------- -------

1 X TBE buffer * ------ ------- -------

Agarose (powder) ------ ------- -------

6 X Loading Dye * ------ ------- -------

Electrophoresis apparatus ------ ------- -------

Casting tray and comb ------ ------- -------

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 thegel. 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.


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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.


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Solutions

Formamide-TE bufferAdd 300 l formamide to 100 l 1x TE

1M Guanidine-HCLDissolve 4.77 g Guanidine-HCL in 50 mL of filtered, distilled deionized water.

10 mg/mL Ethidium bromide recipeDissolve 1.0 g Ethidium bromide in 100 mL filtered, distilled deionized waterWhen dissolved, wrap container in aluminum foil and keep in the dark. Use a dark glass bottle, ifpossible.

6 X Gel loading buffer with glycerol recipe25.0 mg Bromophenol blue25.0 mg Xylene cyanol FF3.0 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, add54.0 g Tris base27.5 g Boric acid20.0 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, add1.0 g NaOH

108.0 g Tris Base55.0 g Boric Acid7.4 g EDTA (disodium salt)Stir to dissolve.Bring up final volume of 1 L.Autoclave to sterilize.

1X TETo 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 mLDissolve 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 8.012.1g Tris base (MW 121.14) in 100mL H2O (adjust pH with concentrated HCl)


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Total RNA Extraction With TRIZOL Reagent 10172007