Conventional RT-PCRs do not allow for discrimination between the sense and anti-sense sequences when used to detect RNAs encoded by overlapping genes. This protocol makes it possible to detect specifically anti-sense RNAs. The use of a biotynylated primers along with high RNA denaturation temperatures allows for amplification of anti-sense cDNAs, preventing the formation of non-specific RT products.
We have used this method to demonstrate that ASP either as an RNA anti-sense or a protein precursor, it's a new HIV antigen, does it potentially a novel HIV target for therapy. This method can be applied to any system, in which anti-sense RNAs from overlapping genes can be detected. Start by designing patient specific primers using the pro-viral DNA sequence internal to the PanASP primers.
Next, quantify the RNA and eliminate DNA contamination by treating samples with DNase. Transfer between 0.1 and one microgram of total RNA into the appropriate wells on a 96 well PCR plate. Then set up the reverse transcription, or RT reaction, according to manuscript directions.
Prepare the endogenous RT controls by adding five microliters of nuclease free water instead of the ASP reverse primer. Put the plate into the thermocycler and heat the RNA to 94 degrees Celsius for five minutes. Then immediately cool it in iced water for at least one minute.
Prepare the reaction mixture in a 1.5 milliliter tube as described in the text manuscript, then transfer 17.5 microliters to each of the RNA containing wells. RNA denaturation must be performed at 94 degrees Centigrade. Without total denaturation, syntheses of non-specific products from sense RNA may occur, leading to amplification of non-specific cDNAs.
Mix the contents of the wells gently and incubate the plate at 55 degrees Celsius for 60 minutes. Then, inactivate the reactions by heating the plate to 70 degrees Celsius for 15 minutes. Prepare one liter of 2X washing and binding buffer according to manuscript directions and filter the solution.
Next, prepare 50 milliliters of 1X washing and binding buffer using PCR grade water. Transfer the appropriate number of streptavidin-conjugated magnetic beads to a 1.5 milliliter tube then wash them by adding an equal volume of 2X washing and binding buffer and vortexing the tube for 15 seconds. Place the tube into a magnetic separation rack and incubate it for three minutes at room temperature.
Then, carefully remove the supernatant without disturbing the beads and add the same volume of the 2X washing and binding buffer as the initial volume of the beads. Vortex the beads for 30 seconds and transfer 10 microliters of the suspension to an appropriate number of 1.5 milliliter tubes. Place the tubes in the magnetic separation rack and incubate them for three minutes at room temperature.
Then, discard the supernatant and add 50 microliters of 2X washing and binding buffer. Add 50 microliters of biotynylated cDNA to the corresponding tubes with the beads and incubate them for 30 minutes while gently rotating. After the incubation, place the tubes on the magnetic separation rack for three minutes then wash the beads with 200 microliters of 1X washing and binding buffer, followed by a three minute incubation on the separation rack.
Discard the supernatant and repeat the wash twice. Then, resuspend the beads in 10 microliters of PCR grade water. Prepare an appropriate volume of PCR Master Mix according to the manuscript directions.
Mix it well and quickly spin it down, then transfer 49 microliters per well to a 96 well PCR plate. Carefully vortex the tubes with the purified cDNA for 15 seconds and add one microliter of the suspension to the corresponding wells. Run the PCR and separate the products on a 1%agarose gel.
Cut the bands from the gel and clone the amplified products into a PCR 2.1 plasmid. Then sequence the clones and analyze each sequence. The initial RT reactions were performed with the regular non-biotynylated anti-sense primer and resulted in successful amplification of ASP.
However, a band of the same molecular weight and primer minus controls was also amplified. To bypass this problem, the specific anti-sense primer was labeled with biotyn and the resulting anti-sense cDNA was purified prior to PCR. This made it possible to amplify the ASP sequence with greatly reduced contamination from the non-specific cDNA in the primer minus controls.
Further optimization of this method was achieved by complete denaturation of RNA at 94 degrees Celsius prior to RT, followed by immediate cooling on ice water, which resulted in effective amplification of the ASP band and no non-specific products. The kinetics of ASP RNA were measured and CD4 cells isolated from three HIV positive subjects with detectable viremia and an absence of therapy following stimulation with anti-CD3/CD28. Quantification of ASP RNA was also possible in patients undergoing ART, with non-detectable viremia.
In two out of three patients, ASP RNA was detected in low levels at three to five days post stimulation. Two patients were analyzed for ASP and env RNAs, one untreated and one treated. For the untreated patient, both RNAs could be detected.
For the treated patient, ASP and env were barely detectable after several days of stimulation. When attempting this procedure, it is important to remember that denaturation linearizes RNA so that secondary structures able to prime RT are destroyed. Whereas, washing of the beads flushes away non-biotynylated cDNAs.
After purification of the cDNAs with the correct polarity, qPCR can be performed to analyze gene expression. Moreover, cDNAs can also be cloned and used for sequence analysis. This method allows studying genes overlapping in opposite orientation and may be useful in elucidating regulation and pathogenesis of a bacteria and a virus-related diseases including some types of cancer.
