The RETROscript® First Strand Synthesis Kit provides reagents for reverse transcription of cDNA from RNA templates. Total RNA, poly(A) RNA, tRNA, or rRNA from a variety of sources (e.g. bacterial, plant , yeast) can be used as a template for reverse transcription. With RETROscript, it is usually not necessary to isolate polyadenylated RNA, even for subsequent amplification of rare messages. Figure 1 demonstrates the amplification of 4 different RNA species from heterogeneous total RNA samples.

Figure 1. RT-PCR of Four Different RNA Species Using Decamer or Oligo dT Primers. Ambion's Total RNAs from several mouse tissues were RT-PCR amplified using Ambion's RETROscript® Kit. Random decamers or oligo dT primers, supplied in the kit, were used to prime the reverse transcription reaction. Primers specific to each of the four target messages were used in the subsequent PCR amplification. Total RNAs used were as follows: Mouse testes RNA for p53, mouse heart RNA for CAP Binding Protein, mouse liver RNA for ß-actin and S15 Ribosomal Protein.

In RETROscript, the Moloney-Murine Leukemia Virus (M-MLV) Reverse Transcriptase is used to extend a random decamer, oligo(dT) or specific primer hybridized to an RNA sample containing the message of interest. M-MLV was chosen for its higher stability and lower intrinsic RNase H activity, compared to AMV Reverse Transcriptase. Both random decamers and oligo(dT) are provided in the RETROscript Kit.

Figure 1 demonstrates the use of each of these primers to amplify four different mRNA species. Note that one or the other type of primer may be better for maximizing yield of an amplified mRNA depending on the specific target (e.g. compare amplification of ß-actin versus S15). A pair of user-supplied, gene-specific primers is then used to amplify the target region via PCR.

The RETROscript Kit provides protocols and reagents for performing the reverse transcription and PCR amplification reactions sequentially (two-step) or as a single reaction. The kit includes two alternative buffers for the first strand synthesis:10X RT Buffer and 10X PCR Buffer. RT reactions using either of these buffers typically yield equal amounts of RT-PCR products. It is important, however, to use the 10X PCR Buffer for the reverse transcription of one-step RT-PCRs. In traditional two-step RT-PCRs, the 10X RT Buffer often gives a higher yield of cDNA than 10X PCR Buffer, but this is not reflected in the amount of product after the PCR step.

A positive control target and primers are included for troubleshooting the kit reagents and procedure. A comprehensive Instruction Manual provides background information as well as protocols for related procedures. The M-MLV and dNTPs included in the RETROscript Kit are also available separately.

Amplification After Reverse Transcription

A thermostable DNA polymerase (not provided in the RETROscript Kit) is required for subsequent amplification of cDNA templates into double-stranded DNA products. Ambion recommends SuperTaq® or SuperTaq® Plus. These high purity Taq DNA polymerases catalyze DNA polymerization in the 5' to 3' direction during repeated heat denaturation and reannealing steps when in the presence of dNTPs, a DNA template and complementary primers. SuperTaq Polymerase can be used in PCR and RT-PCR reactions, and has been optimized for use with Ambion's RETROscript and FirstChoice® RLM-RACE Kits. SuperTaq Plus is an extended range Taq polymerase containing a proofreading activity that reduces the error rate of Taq polymerase. SuperTaq Plus produces significantly higher yields of PCR products than ordinary Taq polymerase, especially for fragments >1 kb, and can amplify up to 20 kb. 10X Reaction Buffer, dNTPs and a MgCl 2 solution are supplied with both enzymes.