Reverse transcription coupled to the polymerase chain reaction (RT-PCR) is commonly used to detect the presence of mRNAs, pre-mRNAs, or other types of RNA such as noncoding RNAs. The method involves using a primer annealed to the RNA of interest. For mRNA, the primer is usually a synthetic oligo(dT)15-18, a random hexamer mixture (dN)6, or a synthetic DNA oligonucleotide that is complementary to a specific transcript (a gene-specific primer). This DNA:RNA hybrid serves as a template during reverse transcription, in which the enzyme reverse transcriptase (RT) generates a single-stranded cDNA copy of a portion of the target RNA molecule. Using random hexamer priming, it is possible to obtain representative cDNA copies of sequences from the entire length of the mRNAs and pre-mRNAs in a population. This cDNA can then be used as a template for PCR. On addition of gene-specific primers, a specific DNA fragment corresponding to a portion of the RNA of interest is generated.
[1]
S Rozen,et al.
Primer3 on the WWW for general users and for biologist programmers.
,
2000,
Methods in molecular biology.
[2]
L. E. Hammond,et al.
Mutations in the hrp48 gene, which encodes a Drosophila heterogeneous nuclear ribonucleoprotein particle protein, cause lethality and developmental defects and affect P-element third-intron splicing in vivo
,
1997,
Molecular and cellular biology.
[3]
J. Sambrook,et al.
Molecular Cloning: A Laboratory Manual
,
2001
.
[4]
Eric E Schadt,et al.
Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing
,
2003,
Genome Biology.