RNA Priming Strategies
First-strand cDNA synthesis reactions can use a combination sequence-specific primers (SSPs), oligo(dT) or random primers. The RNA purification method in combination with the RNA priming strategy chosen can play an important role in cDNA synthesis efficiency, consistency and cDNA yields. While each primer type has its benefits and drawbacks, individual target RNA may respond differently to one primer choice over another. Therefore, each primer option should ideally be evaluated to determine which provides optimal sensitivity and accuracy of first-strand cDNA synthesis.
Sequence Specific Primers
SSPs offer the greatest specificity and have been shown to be the most consistent of the primer options for reverse transcription. However, they do not offer the flexibility of oligo(dT) and random primers, meaning that a new cDNA synthesis reaction must be performed for each gene to be studied. This makes sequence-specific primers less than optimal for processing limiting tissue or cell samples.
One-step RT-PCR reactions always employ a gene-specific primer for first-strand cDNA synthesis, while two-step RT-PCR reactions allow for other priming options.
Oligo(dT) primers are a favorite choice for two-step cDNA synthesis reactions because of their specificity for mRNA and because they allow many different targets to be studied from the same cDNA pool. However, because they always initiate reverse transcription at the 3´ end of the transcript, difficult secondary structure may lead to incomplete cDNA synthesis. Oligo(dT) priming of fragmented RNA, such as that isolated from FFPE samples, may also be problematic. Nonetheless, as long as the primers are designed near the 3´ end of the target, premature termination downstream of this location is less of an issue.
Multiple types of oligo(dT) primers are available. Oligo(dT)20 is a homogenous mixture of 20-mer thymidines, while oligo(dT)12–18 is a mixture of 12-mer to 18-mer thymidines. Lastly, anchored oligo(dT) primers are designed to avoid polyA slippage by ensuring that they anneal at the 3´UTR/polyA junction. Choosing the best oligo(dT) primer may depend in part on the temperature of the reverse transcription. More thermostable RTs such as SuperScript® III Reverse Transcriptase may perform better with longer primers, which remain more tightly annealed at elevated temperatures compared to their shorter counterparts.
Oligo(dT) primers are not recommended as the only primer for cDNA synthesis if 18S rRNA is used for normalization in a real-time PCR experiment as the oligo(dt) primer will not anneal.
Random primers are great for synthesizing large pools of cDNA. They are also ideal for non-polyadenylated RNA, such as bacterial RNA, because they anneal throughout the target molecule. Degraded transcripts such as FFPE samples and secondary structure within the RNA do not pose as big a problem with random primers as they do with gene-specific primers and oligo(dT) primers.
While increased cDNA yield is a benefit, data has shown that random primers can overestimate copy number when used in real-time RT-PCR experiments. Employing a combination of random and oligo(dT) primers can sometimes increase data quality by combining the benefits of both if used in the same first-strand cDNA synthesis reaction. Random primers are used only in two-step qRT-PCR reactions.