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RNA isolation and reverse transcription

Find out the procedure for RNA isolation, DNase treatment and reverse transcription in our detailed protocol for cells in culture and tissue samples.

Procedure for the isolation, DNase treatment and reverse transcription of RNA from cell culture and tissue samples.

Reagents

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Procedure for cells

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1

Stage 1 - RNA isolation procedure for cells and tissue

Aspirate the media using at least 106 cells 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 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 mins.

Centrifuge at the maximum speed (~12,000 x g) for 10 mins.

If a low yield is expected, centrifuge for 30 mins.

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.

This is a critical step; if the pellets dry out too much, the RNA crystallizes and is very difficult to resolubilize. If not enough of the ethanol evaporates, this also prevents the RNA from going into the solution.

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 only difference from the procedure in cells is steps 1-3.

Add 1 mL TRIzol to a sterile culture tube (preferably 12x75 mm) followed by the frozen tissue.

Try not to add more than approximately 20 mg of frozen tissue.

On ice, pulverize the tissue with a homogenizer at a setting of 25 out of 30 for a total of 2 X 10 secs.

Pour the TRIzol solution into a 1.5 mL Eppendorf tube.

Switch over to the 'Procedure for cells' using the button above, and continue from Step 4.

Stage 2 - DNase treatment of RNA samples

Materials required

The DNase cocktail consists of the following (per sample):

Steps

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.

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 mins.

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.

To the remaining RT master mix, add 2 µL of MMLV RT/sample being prepared.

Randomly select two or three samples from a group.

Incubate all samples at 37°C for 1 hr, then 95°C for 5 mins.

Use immediately for PCR or store at -20°C first thing the next day.

Once made, aliquot the two no-RT samples (28 µL each):

The latter step fully inactivates remaining enzymes (i.e. MMLV RT and any remaining DNase).