When your starting material for qPCR is RNA you must first transcribe it into complementary DNA (cDNA) by reverse transcription from total RNA or messenger RNA (mRNA) also known as RT-qPCR.
RT-qPCR can be performed in a one-step or a two-step assay:
One-step assays combine reverse transcription and qPCR in a single tube whereas in a two-step assay, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies.
Click here to the see Advantages and Disadvantages when using one-step versus two-step assays in RT-qPCR
A crucial step for getting your best cDNA strand is primers selection.
Three different approaches can be used for priming cDNA reactions in two-step assays: oligo(dT) primers, random primers, or sequence specific primers.
So, how do we choose? Here are some guidelines
(for more information click here or here):
- Oligo(dT) primers are the optimal choice for constructing cDNA libraries from eukaryotic mRNAs, full- length cDNA cloning, and 3′ rapid amplification of cDNA ends (3′ RACE). However, these primers are not suitable for degraded RNA, nor for RNAs that lack poly(A) tails, such as prokaryotic RNAs and microRNAs.
- Random primers used for RNAs without poly(A) tails, degraded RNA, and RNA with known secondary structures. They are not suitable for full-length reverse transcription of long RNA. A mixture of oligo(dT) and random primers is often used in two-step RT-PCR to achieve the benefits of each primer type.
- Gene-specific primers are designed based on known sequences of the target RNA. Since the primers bind to specific RNA sequences, a new set of gene-specific primers is needed for each target RNA. As a result, more RNA is required for analysis of multiple target RNAs. Gene-specific primers are commonly used in one-step RT-PCR applications
Reverse transcriptases – a family of enzymes:
Reverse transcriptases are a broad family of enzymes. The right choice can significantly affect your research.
Some enzymes have RNase H activity to degrade the RNA strand in the RNA-DNA hybrid after transcription. Reverse transcriptases with intrinsic RNase H activity are often favored in qPCR applications because they enhance the melting of RNA-DNA duplex during the first cycles of PCR.
For difficult samples in RT-qPCR, it is ideal to choose a reverse transcriptase with higher thermal stability, because this allows cDNA synthesis to be performed at higher temperatures, ensuring successful transcription of RNA with high levels of secondary structure, while maintaining their full activity throughout the reaction producing higher cDNA yields.
Here are common reverse transcriptases and their attributes:
AMV reverse transcriptase1
MMLV reverse transcriptase2
Engineered MMLV reverse transcriptase
RNase H activity
(with challenging or suboptimal RNA)
Derived from the pol gene of:
1 Avian myeloblastosis virus (AMV)
2 Moloney murine leukemia virus (MMLV)
Click here and choose which kit is best for your needs