The overall goal of this procedure is to determine the sequences of mRNA transcripts from the three-prime end to regions within the protein coding region by using transcript-specific nested primers in two subsequent PCRs and sequencing the purified PCR products. This method can help answer key questions in the genetic field, such as determining the polyadenylation signal, the stop codon, and the sequence of the three-prime untranslated region of the transcript. The main advantage of this technique is that it can be applied to any transcript if the sequence of the small region of the open reading frame is The implication of this technique extend toward cancer diagnosis, because it can detect tumor specific fusion genes and splice variants.
We first had the idea for this method when we identified novel transcripts with previously uncharacterized, three-primed untranslated regions. To begin the protocol, thaw the phenol and guanidine isothiocyanate cell mixture on ice. Once thawed, add 100 microliters of chloroform to the microcentrifuge tube in a PCR hood.
Vortex the sample for 10 to 15 seconds until the mixture is pink and opaque. Next, incubate the sample on ice for 15 minutes. After incubation, centrifuge the sample for 15 minutes at 20, 800 g and four degrees Celsius.
Carefully remove the upper colorless aqueous phase and transfer it to a new 1.5 milliliter microcentrifuge tube. Then, move on to precipitation and add an equal volume of isopropanol to the sample. Add one microliter of co-precipitant to the sample to act as a carrier for the RNA and to help visualize the RNA in subsequent steps.
Incubate the samples at 80 degrees Celsius for at least four hours. After incubation, transfer the sample to a cooled centrifuge and spin for 35 minutes at 20, 800 g and four degrees Celsius. After the spin, the RNA will appear as a blue spec on the bottom of the tube.
Carefully remove the isopropanol from the sample. Add 500 microliters of 80%ethanol and resuspend the pellet by briefly vortexing for three seconds. Centrifuge the sample again.
Remove all the ethanol from the sample and let the sample air dry for about 10 minutes. Once the sample is dry, resuspend it in 35 microliters of RNase-free water. Then, place the sample on a heat block.
Set at 65 degrees Celsius for five minutes to ensure that the RNA is fully in solution. After heating, immediately place the tube on ice. For a final reaction volume of 50 microliters, add four micrograms of total RNA with added water to a total volume of 22 microliters to a new tube.
Add two microliters of the Oligo dT 25 primer from the 10 micromolar primer solution. From the reverse transcription kit, add two microliters of the RNase inhibitor, eight microliters of the 5x reaction buffer, four microliters of 10 millimolar dNTPs, and two microliters of the reverse transcriptase. Set up a reaction without reverse transcriptase to act as a negative control.
Incubate the tube at 42 degrees Celsius for one hour. After incubation, transfer the tube directly to ice before transporting it to the heating block. Then, heat the tube at 75 degrees Celsius for five minutes.
After heating it 75 degrees Celsius, place the tube on ice. Transfer two microliters of the cDNA to a fresh 0.5 milliliter PCR tube. Add one microliter of the forward primer and one microliter of the reverse primer from a 10 millimolar primer solution.
Make up to 12.5 microliters by adding 8.5 microliters of nuclease-free water and add 12.5 microliters of the 2x PCR to gel master mix. Then, enter the PCR thermal cycling conditions listed on the screen and run the PCR. After the PCR, prepare a 1%agarose gel stained with ethidium bromide.
Dissolve one gram of agarose in 100 milliliters of triacetate or TAE buffer in a 250 milliliter flask. Boil the contents in a microwave. Allow the gel to cool for one minute, and add 7.5 microliters of ethidium bromide solution in a fume hood.
Mix well by swirling the flask. Pour the contents into a horizontal gel apparatus and allow the gel to solidify at room temperature in the fume hood for at least 30 minutes. Cover the gel with 1x TAE buffer and load 10 microliters of the PCR product onto the agarose gel together with three to five microliters of the DNA molecular weight ladder.
Run the gel at 175 volts for 10 minutes. For the first PCR, transfer one microliter of cDNA to a PCR tube and add five microliters of 10x Pfu reaction buffer. Add one microliter of the first nested forward primer, one microliter of the T7 Oligo dT 25 primer, and one microliter of dNTPs from a 10 millimolar solution.
Make up to 49 microliters of total volume by adding 40 microliters of water. Add one microliter of Pfu DNA polymerase and mix well. Then, run the PCR.
Next, prepare the second PCR. Transfer two microliters of the products from the first PCR to a new PCR tube and mix it with five microliters of 10x Pfu ultra two reaction buffer. Add one microliter of the second nested PCR primer, one microliter of the T7 primer and one microliter of dNTPs.
Make up to 49 microliters total reaction volume by adding 39 microliters of water and add one microliter of Pfu DNA polymerase. Run the PCR using the same PCR profile as the first PCR set up. Alternatively, instead of using Pfu DNA polymerase, a chimeric DNA polymerase can be used in the two different PCRs.
Take five to ten microliters of the second PCR product and run it on an agarose gel. Finally, visualize the result on an imager. This agarose gel shows two distinct PCR products which use the same forward primer but different reverse primers and a distinct PCR product that has a distinct forward and reverse primer.
The ideal primers to use for the PCR based reaction are those that give one distinct PCR product. Products from the second PCR reaction with Pfu DNA polymerase and chimeric DNA polymerase produced the same sized PCR products despite having different PCR cycling conditions for the three-prime RACE. The reverse transcriptase negative controls showed no genomic contamination of the RNA used in cDNA synthesis as well as in subsequent downstream reactions.
Gel-purified PCR products were sent for sequencing. A representative Sanger sequence from a sequence chromatogram identified the location of the stop codon, putative polyadenylation signal and the site as well as the poly A tail in the three-prime RACE product. While attempting this procedure, it's important to remember to wear protective gear and gloves at all time.
In addition, use sterile DNase and RNase for your reagents tubes and other disposables. Don't forget that working with phenol and chloroform can be extremely hazardous. And precautions such as using the reagents in a hood and disposing material in a designated waste container should always be taken while performing this procedure.
