Effects of primers with different properties on the reverse transcription process

① Oligo(dT)

Oligo(dT) primer is a repetitive oligonucleotide sequence consisting of about 12-18 polythymine Ts, which can specifically anneal to the ploy(A) tail of eukaryotic mRNA, so it is not suitable for lack of ploy(A) RNAs with tail structures, such as prokaryotes, RNA or microRNA, are also not suitable for degraded RNAs, such as RNA in FFPE samples.

mRNA in eukaryotes only accounts for about 1%-5% of total RNA, and Oligo(dT) primers are usually preferred when constructing cDNA libraries from eukaryotes, or when cloning full-length cDNA and 3'RACE.

For complex template RNAs, such as those with more secondary structures, in the process of cDNA synthesis and extension, when extending to the secondary structure, the extension may be terminated. If Oligo(dT) is selected, it may cause the 5' end of mRNA loss of information.

② Random N6-N9

Random primers are oligonucleotides consisting of random bases, usually 6 nucleotides, called random hexamers. The primers do not have specificity, and rRNA, tRNA, non-coding RNA, small RNA, degraded RNA, RNA with complex secondary structure, etc. can be used as their templates. Such primers can increase the yield and concentration of cDNA, but will shorten the length of the synthesized cDNA.

③ Gene-specific primers

Gene-specific primers can be specifically complementary to templates, and generate highly targeted cDNA under the action of reverse transcriptase. They are more specific for subsequent PCR amplification and are suitable for known target sequences. Usually used in one-step RT-PCR reactions. When the first-strand cDNA cannot be efficiently synthesized using gene-specific primers (GSP), Oligo(dT) or random primers can be used.

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