One of the many facets of life science research that makes it so interesting is the cyclic nature of the techniques we use over time. We may spend a few weeks subcloning various inserts, running immunoblots, or analyzing microarray data. My current cycle involves quite a bit of reverse transcription. One project involves making a phagemid library from lymphocyte mRNA and another is focused on RT-PCR of a particular transcript in numerous osteosarcoma specimens. Because my institution has a Roche Depot onsite, I started using their Transcriptor Reverse Transcriptase (Transcriptor RT, Roche Applied Science, Indianapolis, IN) at the beginning of the projects and have been using it ever since.
Transcriptor RT is a new recombinant reverse transcriptase with a number of useful features. The enzyme accepts both RNA and DNA as template, possesses unwinding and RNase H activity, and is stabile at temperatures up to 65C. The unwinding activity helps the polymerase plow through regions of secondary structure and RNase H activity obviates the need for an extra incubation step to degrade the RNA in RNA:DNA hybrids. The ability to carry out the reaction at any temperature from 55C to 65C provides sufficient flexibility to handle transcripts of varying GC content.
Transcriptor RT can be purchased alone, in package sizes of 250, 500, or 2000 u, or as a kit. Both choices include 5X Reaction Buffer but the kit also includes Protector RNase Inhibitor, dNTPs, an anchored oligo (dT)18 primer, a random hexamer primer, control RNA, forward and reverse primers for the house-keeping gene porphobilinogen deaminase (PBGD), and PCR-grade water. I’ve been buying the RT alone and purchasing the RNase inhibitor, dNTPs, and an oligo (dT)15 primer separately. If you add in all of these costs, buying the components separately costs about the same as the kit on a per reaction basis. Using either the kit or purchasing the components separately costs around $5.50/rxn. Although it hasn’t been a problem in my projects, the anchored oligo (dT)18 primer may be better in some instances as it has been designed to bind at the beginning of the poly(A) tail and increase the proportion of full-length transcripts.
The recommended protocol is quite simple. Basically, you combine the mRNA or total RNA template with primer, add water to 13 ul and then heat at 65C for 10 min to melt any secondary structure. The heated mix is then put on ice until the addition of the remaining ingredients, which consist of 5X RT buffer, dNTPs, RNase inhibitor, and RT. The recommended incubation is 30 min at 55C. Alternatively, you can incubate at a higher temperature, up to 65C, for GC-rich templates. The RT can be heat inactivated by incubating at 85C for 5 min. The resulting cDNA can be used directly for PCR. I routinely use 2 ul for a 25 ul reaction. Roche recommends 1-5 ul for a 50 ul reaction. So far, Transcriptor RT has worked beautifully on a wide range of starting RNA concentrations. I generally shoot for 1 ug total RNA but have used down to 80 ng with good results.
I used to use a one-step RT-PCR kit but prefer a 2-step procedure. With the 2-step protocol, you can freeze the cDNA and use it whenever in a simple PCR reaction. The cDNA is more stable than RNA so it’s easier to store.
The protocol also makes it simple to deal with very dilute RNA samples. I usually take a volume of the dilute solution sufficient to give me 1-2 ug total RNA and precipitate it with ethanol/sodium acetate. After washing the pellet in 70% ethanol and air drying it, I add water and oligo (dT) primer directly to the pellet. The 10 min incubation at 65C solubilizes the RNA very efficiently and denatures it at the same time. After cooling on ice, it’s ready for reverse transcription.
In summary, I have found Transcriptor RT from Roche to be a very solid product. It is reasonably priced, easy to use, and reliable. I highly recommend it.
Michael Campa, Ph.D.
Assoc. Research Professor of Radiology
Duke University Medical Center