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 0.1 M, primers with 0.1 μg/μL or 1/30 dilution of 3 μg/μL, BSA

tabName

Procedure for cells

timelineNumber

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

• 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.

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.

Place samples on ice

• Then pour off the isopropanol and add 1 mL 75% ethanol in DEPC-treated H₂O.
• Mix gently.
• Recentrifuge at 11,500 x g for 5 minutes.

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.

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 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.

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):

• 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.

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.

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

• 13 µL DEPC treated water
• 16 µL 5X first strand buffer
• 7 µL DTT (0.1 M)
• 8 µL random primers (concentration = 0.1 µg/µL or a 1/30 dilution of the 3 µg/µL)
• 8 µL BSA
• 3 µL dNTPs​
• 1 µL RNase Out

Steps

For each sample, mix together the reagents by vortexing.

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.

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.

• Mix by vortexing, and then aliquot 30 µL per tube for each sample
• For example, if you have a total of 10 samples, you will reverse transcribe all 10, but want 2 or 3 samples additionally for no RT controls to assure that the DNase treatment step worked. Thus, you would multiply the above master mix by 7 or 8. 1X the master mix is for two samples.

Randomly select two or three samples from a group.

• Prepare no-RT controls by aliquoting out 28 µL per sample into 0.5 mL tubes
• Add 1 µg of RNA to each along with an additional 2 µL of DEPC-treated H₂O (this is optional)

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):

• Add 20 µL of the MMLV RT (2 µL/sample) to the master mix.
• Vortex the mix, then aliquot 30 µL per tube for 10 tubes.
• To each, add the corresponding 1 µg (10 µL) of RNA and incubate.