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RNA isolation and reverse transcription
Procedure for the isolation, DNase treatment and reverse transcription of RNA from cell culture. and tissue samples.
Reagents
- Ice-cold PBS
- TRIzol
- Chloroform
- Isopropanol
- 75% ethanol
- DEPC treated H2O/water
- DEPC treated TE buffer
- DNase cocktail: Rq1 RNase free DNase, DNase 10X reaction buffer, DEPC-treated H2O, RNase Out
- RT sample: DEPC-treated water, 5X first stand buffer, DTT 0.1M, primers with 0.1ug/ul or 1/30 dilution of 3 ug/ul, BSA
Stage 1 - RNA isolation procedure for cells and tissue
Steps
Aspirate the media
Using at least 106 cells, aspirate the media and wash once with ice-cold PBS (1–2 mL).
Aspirate the PBS (remove as much as possible) and add 1 mL TRIzol.
Scrape the plate briefly, then remove the TRIzol with a pipette and deposit the TRIzol/cell lysate into a 1.5 mL Eppendorf tube.
Leave at room temperature for 5 min
Add chloroform
Add 250 µl chloroform and shake the tube vigorously for about 15 sec.
Leave at room temperature for 5 min
Centrifuge at 10,000 rpm for 5 min
Carefully remove the aqueous phase using a pipette.
At this point, there will be three layers in each tube:
Top layer: clear, aqueous
Middle layer/interphase: white precipitated DNA
Bottom layer: pink organic phase
- Carefully remove the aqueous phase using a pipette.
- Leave behind some of the aqueous phase (about 1 mm above DNA layer to prevent DNA contamination).
- Place in another 1.5 mL Eppendorf tube.
Avoid working with large pipettes here, as it is harder to control the rate and force of fluid withdrawal and this increases the likelihood of drawing some of the organic or DNA phase.
Add 550 µl isopropanol to the aqueous phase and mix gently.
Leave at room temperature for 5 min
Centrifuge at maximal speed (14,000 rpm) for 20 min
If a low yield is expected, centrifuge for 30 min
Place samples on ice
- Then pour off the isopropanol and add 1 mL 75% ethanol in DEPC-treated H2O.
- Mix gently.
- Recentrifuge at 9,500 rpm for 5 min.
There should be a pellet barely visible at the base of each tube.
Pour off the ethanol and let the pellets air dry
Centrifuge the tubes to quicken the evaporation
- After pouring off the bulk of the ethanol wash, there will be approximately 30–40 µl left in the bottom of the Eppendorf tube.
- To quicken the evaporation, centrifuge the tubes briefly to force the remaining fluid on the side of the tube to the bottom, then pipette off as much of the ethanol as is feasible.
The best time to add DEPC-treated water to the RNA pellet is when there is only a tiny meniscus of solution left around the pellet itself.
Add water to the RNA pellet
- Add approximately 15–25 µl (depending on yield) of either DEPC-treated TE buffer or water to the RNA pellet.
- To a small Eppendorf tube, dilute the RNA 1/40 (1.2 µl in 48.8 µl of TE buffer) and add to a microcuvette (path length = 1 cm).
- Then measure the absorbance at 260 nm.
The 260/280 ratio should be greater than 1.8. If less than 1.5–1.6 or so, the RNA is likely to be at least partially degraded. Lower ratios also suggest DNA or thiocyanate contamination. The concentration is essentially the equivalent of the OD at 260 nm (in µg/µl).
Stage 2 - DNase treatment of RNA samples
Materials required
The DNase cocktail consists of the following (per sample):
- RQ1 RNase free DNase: 1 µl
- DNase 10x reaction buffer: 2 µl
- DEPC-treated H2O: 6 µl
- RNase Out: 0.5 µl
Steps
Make a master mix of the above based on the number of RNA samples being treated.
Prepare the RNA in the following way
- Add 2 µg of RNA (calculated by 2 µg/the concentration in µg/µl) to a small Eppendorf tube.
- Bring the total volume of the RNA to 11 µl by adding additional DEPC-treated water.
For example, if your RNA concentration is 1 µg/µl, add 2 µl of the RNA to 9 µl of DEPC water.
Add 9 µl of the DNase master mix to the RNA bringing the total volume to 20 µl
Using a thermal cycler, incubate the samples at 37°C for 15 min, followed by 65°C for 20 min, then place on ice.
Briefly centrifuge each sample to assure all of the volume lies in the bottom of the tube
The DNase-treated RNA can then be used immediately for the reverse transcription reaction.
Stage 3 - Reverse transcription of DNase treated RNA
Under most circumstances, each sample of RNA (1 µg, or 10 µl from the DNase treatment reaction) will be run with reverse transcriptase with the second 1 µg aliquot being used for a no-RT control.
Materials required
Steps
For each sample, mix together the reagents by vortexing
The total for each sample should be 56 µl
Aliquot 28 µl each into two separate 0.5 mL Eppendorf tubes
- One tube will serve as the RT reaction (to which 2 µl of the MMLV reverse transcriptase is added) and the other, the no-RT control, to which 2 µl of H2O is added.
To each tube, add 10 µl of the DNase treated RNA from above
Mix well by pipetting
Incubate all samples at 37°C for 1 hr, then 95°C for 5 min
The latter step fully inactivates remaining enzymes (i.e. MMLV RT and any remaining DNase).
Use immediately for PCR or store at -20°C first thing the next day