We assessed the PCR-primer ratio for synthesis of single stranded DNA in the in situ RT-PCR indirect. We tested 4 different primer ratio (sense versus antisense), 50:1, 50:0.2, 50:0.1 or 50:0.05 for detecting rat insulin mRNA. To detect the single stranded DNA synthesized in the RT-PCR, we used the specific probe in three different concentrations, 4, 2 or 1 μg/ml. The intensity of the detection was dependent on the concentration, strong positive by 4.0 μg/ml probe, intermediate positive by 2.0 μg/ml and weak positive by 1.0 μg/ml. The detection level of the control products, which were produced using primers whose ratio were 50:50 or 50:0, were weak positive by 2.0 μg/ml probe and negative by 1.0μg/ml. The results indicated that the primer ratio of 50:1 to 0.05 gave the sufficient intensity in the hybridization. Single stranded DNA in the specimen synthesized prior RT-PCR makes the hybridization supersensitive.
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