Procedure for the isolation, DNase treatment and reverse transcription of RNA from cell culture. and tissue samples.
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 12,000 x g for 15 minutes
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
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 the maximum speed (~12,000 x g) for 10 min
If a low yield is expected, centrifuge for 30 min
Place samples on ice
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
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
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).
The DNase cocktail consists of the following (per sample):
Make a master mix of the above based on the number of RNA samples being treated.
Prepare the RNA in the following way
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.
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.
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
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