RETROscript™ First Strand Synthesis Kit For RT-PCR From Ambion

Ambion considers themselves “The RNA Company” and rightfully so, because every product from this company has proven itself in my lab. This Austin, TX based company is a leader in products for molecular biology and RNA-based research. They offer all sorts of products from those which are used in isolating and detecting RNA to products for synthesizing and measuring RNA. Almost anything that you could think of that is required for RNA manipulation, this company can provide. The website for this company also contains excellent technical resources on almost any molecular biology question you might have. The RETROscript™ Kit has been an invaluable tool in my lab and our research on the response of numerous genes to various toxins. It has provided consistent results over the 2 years I have used it.

The RETROscript™ Kit contains all the components necessary to copy RNA into its complementary DNA sequence (cDNA) except the thermocycler, disposable tubes, pipettes, and 1-2 ug of template RNA. The kit also offers flexibility and options which allow the individual researchers to adapt it to their own experiments. It allows for 40 reverse transcription reactions and 40 PCR reactions. The complete kit contains the 4 deoxynucleotide triphosphates (dNTPs) premixed for ease-of-use, 10X Reverse Transcriptase (RT) and PCR buffers, nuclease free water, Moloney-murine leukemia virus Reverse Transcriptase (MMLV-RT), random decamers, oligo(dT) primers, and RNase inhibitor. The kit also includes control PCR primers, control template RNA, and a high resolution gel loading buffer. The template used with this kit can be total RNA, poly(A) RNA, tRNA, or rRNA that is extracted from a number of sources ranging from bacterial to mammalian cells.

You have several options (all listed in the manual) on how to set up your experiment. You can either do a 1-step or 2-step RT-PCR. The 1-step reaction is performed by combining all the reagents needed for both reverse transcription and PCR into one tube. This allows you to save time by not having to set up a separate PCR reaction once the cDNA is made during the RT step. I have not performed the 1-step reaction so I cannot comment on whether this is a reliable method. The 2-step method is the one I use consistently and I know to be reliable. This method involves reverse transcribing the RNA into cDNA first. Then, in a separate reaction, the PCR on the gene of choice is performed. Each time I have performed this method, I used RNA I have extracted from either mammalian cells or tissue using the Tri Reagent. Then, I typically clean the RNA by following the RNA cleanup protocol from Qiagen’s RNeasy Mini Kit. I also perform the optional on column DNase digestion provided within the Qiagen kit to remove any genomic DNA contamination. This is not necessary, however, as I have used the RETROscript™ Kit on unclean DNA before and the cDNA that was produced was high quality. I have always used the oligo(dT) primers during the RT step, but you also have the option of using random decamers. These primers might be of use if your template is limiting since the final RT-PCR yield might be a little larger when you use those primers instead of the oligo(dT) primers.

The protocol is extremely fast and easy. The whole process of setting up the reactions with RNA to the final product of cDNA takes approximately 1 hour and 45 minutes. This time includes a 3 min incubation at 70ºC, a 1 hour incubation step at 42ºC, and a 10 min incubation step at 92ºC. The 70ºC incubation is for denaturation of any secondary structures; it is recommended, but not required. You have the option of removing this step if you want.

The RETROscript™ Kit has produced excellent quality cDNA for the PCRs performed in my lab on many genes, including COX-2, iNOS, IL-6 and IL-8. This kit is user friendly with a very informative and easy-to-understand instruction manual. The kit may be expensive to many budgets, but it is well worth the price. To save reagents, I would suggest not using the control primers and template they provide unless you are having problems. I never have used their control reagents and have not had an issue. Also, I would not run a control of your own RNA without the reverse transcriptase to check for genomic DNA contamination. This only wastes the RT enzyme which is the limiting reagent for this kit. Instead, as a control, I use the RNA in the PCR reaction. If DNA is synthesized during PCR, then you know that you have genomic DNA in your RNA sample. If nothing is amplified, you can be confident there is no contamination.

Overall, the RETROscript™ Kit from Ambion is an essential item to have in any laboratory performing molecular biology techniques. It has been a reliable and effective tool in our research.