This method can help answer key questions in the RNA biology field, such as what is the biological significance of RdRP or TERT in human cells? The main advantage of this technique is that this assay is established as the first sensitive method to detect human RdRP activity expressed in cells. Begin this procedure with preparation of HeLa cells as described in the text protocol.
Wash the cells once with 10 milliliters of PBS. Then, centrifuge the sample at 1, 450 times gravity for five minutes at four degrees Celsius, and discard the supernatant. Use one milliliter of ice-cold Lysis Buffer A per 10 million cells to lyse the cells by gentle pipetting.
Sonicate the sample in a 1.5-milliliter tube at a sonicator amplification of 25%for 10 seconds. Following sonication, centrifuge the sample at 20, 400 times gravity for 15 minutes at four degrees Celsius. Collect the supernatant, and transfer one milliliter each of the supernatant into 1.5-milliliter microcentrifuge tubes.
Pre-clear the lysate by adding 40 microliters of prewashed Protein A-agarose beads per one milliliter of the lysate. Mix well by inverting the tube several times. Then, rotate the sample for 30 minutes at four degrees Celsius.
Centrifuge the sample at 13, 000 times gravity for one minute at four degrees Celsius, and recover the supernatant. Next, add 40 microliters of prewashed Protein A-agarose beads and 10 micrograms of anti-human TERT antibody into the pre-cleared lysate. Mix well by inverting the tube several times, and rotate the sample at four degrees Celsius overnight.
Following centrifugation at 3, 300 times gravity for 30 seconds at four degrees Celsius, remove the supernatant. Next, wash the beads with Wash Buffer One. Add one milliliter of Wash Buffer One to the beads, and mix well by inverting the tube.
Then, rotate the sample for five minutes at four degrees Celsius. Centrifuge the sample at 3, 300 times gravity for 30 seconds at four degrees Celsius. Remove the supernatant, and repeat this step three additional times.
Following AGC solution wash as described in the text protocol, treat the beads with micrococcal nuclease by resuspending them in 60 microliters of the micrococcal nuclease reaction mixture by gentle pipetting. Then, shake the sample gently on a turntable of a shaker set in a Peltier-type incubator for 15 minutes at 25 degrees Celsius. After centrifuging the sample at 3, 300 times gravity for 30 seconds at four degrees Celsius, remove the supernatant.
Finally, wash the beads twice with an AGC solution containing three millimolar EGTA, and wash the beads once with Wash Buffer Two as described in the text protocol. Prepare the RdRP reaction mixture in a new tube as described in the text protocol. Add six microliters of uridine triphosphate labeled on the alpha phosphate group with P dash 32 to the reaction mixture, and mix well by pipetting.
Then, add one microliter of template RNA to the mixture, and mix well. Resuspend the beads with 20 microliters of the resulting RdRP reaction mixture. Next, shake the sample gently on a turntable of a shaker set in a Peltier-type incubator for two hours at 32 degrees Celsius.
Following incubation, add 5.4 microliters of 20 milligram per milliliter Proteinase K and 45.4 microliters of 2X Proteinase K Buffer to the reaction mixture. Mix by pipetting, and shake the sample in a Peltier-type incubator for 30 minutes at 37 degrees Celsius. After adding 109.2 microliters of RNase-free water to the sample, add 200 microliters of acid phenol-chloroform.
Mix well by vortex before centrifuging the sample at 21, 100 times gravity for five minutes at room temperature. Transfer the aqueous phase to a new tube. Repeat this step once.
Then, add 20 microliters of three molar sodium acetate, pH 5.2, four microliters of precipitation carrier, and 250 microliters of ethanol to the aqueous phase. Shake the tube well for mixing. Centrifuge the sample at 21, 100 times gravity for 20 minutes at four degrees Celsius, and discard the supernatant.
Next, wash the pellet with 300 microliters of cold 70%volume per volume ethanol stored at minus 20 degrees Celsius. Centrifuge the sample at 21, 100 times gravity for 15 minutes at four degrees Celsius. Discard the supernatant, and air-dry the pellet.
Resuspend the pellet in 20 microliters of RNase-free water. Prepare the RNase reaction mixture in a new tube as described in the text protocol. Add 180 microliters of the RNase reaction mixture to the sample.
Then, incubate the tube for two hours at 37 degrees Celsius. Add 2.3 microliters of 10%volume per volume SDS to the sample, and incubate for 15 minutes at 37 degrees Celsius. Then, add two microliters of 20 milligrams per milliliter Proteinase K to the sample, and incubate for another 15 minutes at 37 degrees Celsius.
Now, add 205 microliters of acid phenol-chloroform to the sample. After mixing and centrifuging as before, transfer the aqueous phase to a new tube. Add three molar sodium acetate, precipitation carrier, and ethanol to the aqueous phase, followed by mixing, centrifuging, and washing the pellet as performed earlier.
Shown here are three representative results of the IP-RdRP assay in HeLa cells treated with nocodazole or DMSO for unmanipulated cells. A chemically synthesized 34-nucleotide RNA was used as a template. After overnight exposure, RdRP products can be seen between 20 to 30 nucleotides, specifically in HeLa cells within the mitotic phase.
After watching this video, you should have a good understanding of how to detect the RdRP activity of human TERT.
