When performing a RT it is possible to use three different primer types:
Oligo d(T) primers, which bind to the poly A tail of the RNA and then only transcribe RNA. This will avoid contamination with genomic DNA. As the poly A tail is located at the beginning of the gene it will also lead to more full transcripts.
Ramdom nonamers, which bind anywhere in the genome and allow the reverse transcriptase to fill up the gaps, will leads to high yields.
Specific primers, which bind to the gene of interest, and will therefore give specifics products.
The combination of oligo d(T) primers and random nonamers will give the highest yields and the longest transcripts, whereas specific primers transcribe only specific RNA but reduce the yield.
With an One step RT qPCR kit it is only possible to add specific primers, as it should be avoided that oligo d(T) primers and ramdom nonamers participate in the PCR reaction, giving many aspecific products. As a RT reaction is performed at 40-50°C, the primers can bind with mismatches to the RNA and therefore transcribe unwanted sequences, which then also will be amplified in to consecutive PCR, leading to aspecific PCR products. This disadvantage is inherent to the method.
In a Two step kit the oligo d(T) primers and the ramdom nonamers are included in the kit and will give ride to cDNA. Then two specific primers have to be selected and this will amplify the exact sequence.