Reverse transcription is the reverse transcriptase (RT enzyme)–mediated synthesis of single-stranded DNA (complementary DNA or cDNA) using single-stranded RNA as template. The cDNA can be used as a template for amplification by PCR, or to generate a cDNA library.
Any RNA—total RNA, mRNA, specific RNA, in vitro transcribed RNA—can be reverse transcribed as long as a single-stranded DNA primer is hybridized to the RNA; the reverse transcriptase (RT) enzyme will start synthesizing DNA from the 3' end of the primer using the RNA sequence as template (Figure 1). An oligo(dT) primer will allow cDNA synthesis from almost all mRNAs, as it can bind to the poly(A) tails found on most mRNA molecules. For non-polyadenylated RNA, such as bacterial RNA, random primers are suitable for synthesizing large pools of cDNA because they anneal throughout the target RNA. On the other hand, one can use a gene-specific primer to reverse transcribe specific mRNA. The strategies for RNA priming, in combination with RNA purification methods, play an important role in cDNA synthesis efficiency, consistency, and yield.
Figure 1. Reverse transcription protocol.
Commonly used RT enzymes are viral products (e.g., from avian myeloblastosis (AMV) or Moloney murine leukemia virus (M-MLV). Both the AMV and M-MLV RT enzymes have RNase H activities, which compete with the polymerase activity for the hybrid formed between the RNA template and the DNA primer. This competition decreases the amount of the template-primer complex, resulting in lower yields of cDNA. In addition, the RNase H activity acts to diminish the yield and size of the cDNA by hydrolyzing the RNA template as chain growth occurs. Eliminating the RNase activity is critical to obtaining higher yields of full-length cDNA.
To obtain higher yields of full-length cDNA, reverse transcriptases have been engineered to remove their RNase H activity without affecting their polymerase activity. These engineered RTs, like SuperScript® III, have higher processivity and lower error rates, and they can function at higher temperatures that allow resolution of RNA folds and less nonspecific binding by the DNA primer.
The advantages of SuperScript® III over other RTs are:
- Highest specificity with gene-specific primers, compared to other RTs (Figure 2)
- 55oC reaction temperature, which alleviates secondary structure
- Up to 400 units per reaction without inhibiting subsequent PCR
Figure 2. The SuperScript® One-Step RT-PCR System with Platinum® Taq polymerase. One-step RT-PCR reactions were performed with 0.01 pg, 0.1 pg, 1.0 pg, and 1.0 ng of total HeLa RNA using reagents and conditions specified in each manufacturer's protocol. The annealing temperature was 55°C for all reactions.
Selecting the correct reverse transcriptase for cDNA synthesis is critical to obtaining high yields of high-quality, full-length cDNA. Life Technologies offers a wide range of RTs optimized to generate cDNA for your research needs (Table 1).
Table 1. Features of different reverse transcriptases.
|SuperScript® III||SuperScript® II||M-MLV||AMV|
|Source||Moloney murine leukemia virus||Moloney murine leukemia virus||Moloney murine leukemia virus||Avian myeloblastosis virus|
|Optimal reaction temperature||50–55°C||42°C||37°C||42–62°C|
|Target size||<12.3 kb||<12.3 kb||<7 kb||<12.3 kb|
|RNase H activity||Low||Low||High||Highest|