Complementary DNA-bound RNA was then removed by RNAse H (2 U; GibcoBRL) digestion at 37°C for 20 min. PCR was performed in a 25-^l reaction containing an RT aliquot equivalent to approximately 10% of the RT reaction, 0.2 mM dNTP mix, 0.25-0.5 ^M upstream and downstream primers (each), 0.5 ^Ci [a-32P]CTP (3000 Ci/mmol; New England Nuclear, Boston, MA), and 1.25 U Taq polymerase in single-strength PCR buffer A (both from Fisher Scientific, Pittsburgh, PA). After denaturation at 94°C for 3 min, templates were amplified for 22 cycles using a temperature profile of 94°C, 58°C, and 72°C for 30 sec each in GeneAmp PCR System 9600 (Perkin Elmer, Norwalk, CT). my canadian pharamacy
Five microliters of single-strength reaction mix containing 1.25 U Taq was then added, and PCR was continued for additional 25 cycles. Ten microliters of the PCR product was resolved by electrophoresis on 5% polyacrylamide gels in single-strength Tris-borate-EDTA buffer and then autoradiographed for 1 h at -80°C. Quantitation of gels was performed by beta-counting radioactive gel bands. For each PCR product, an exponential relationship was observed between increasing RNA concentration and PCR product intensity, although for many targets, the conditions used in these studies were on the plateau phase of the amplification curve to maximize sensitivity.