What are the benefits and application of two-step RT-qPCR?
What are the benefits and application of two-step RT-qPCR?
Two-step RT-qPCR allows for the preparation of total cDNA by reverse transcription using universal primers that recognize all mRNA molecules, such as random hexamers or oligo-dT primers. The resulting cDNA can be used for the detection of a variety of transcripts. When there is an overabundance of total RNA in the reaction, oligo-dT primers are recommended for two-step RT-qPCR rather than random 6-mers. The use of oligo-dT primers may improve efficiency and provide similar results as compared to one-step RT-qPCR. When amplifying cDNA produced by oligo-dT primed RT, the PCR amplicon should be located near the 3' end of the expected cDNA. Two-step RT-qPCR is used when analyzing the expresssion of a large number of genes.