Taqman® MicroRNA Reverse Transcription Kit From Applied Biosystems

The TaqMan® MicroRNA Reverse Transcription Kit is specifically designed to generate microRNA specific cDNA, for use with the TaqMan® MicroRNA Assays. These assays are an invaluable tool for scientists interested in the field of microRNA research and allow the user the user to detect and accurately quantify specific mature microRNAs.

A novel new technology called looped-primer reverse transcription (RT) is employed by the TaqMan® reverse transcription kits, allowing the user to reverse transcribe only the mature micoRNA of interest and not its precursor. Within this system, a looped reverse transcription primer targets the mature microRNA of interest within the total RNA sample. This then primes the miRNA for MuLV reverse transcriptase mediated reverse transcription.

The kit is available in 200 and 1000 reaction sizes and includes 100 mM dNTPs, MultiScribe™ reverse transcriptase (50 U/ul), 10X reverse transcription buffer and RNase Inhibitor (20 U/ul). Not included in the kit is nuclease-free water, or the microRNA specific RT primer, which instead is supplied as part of Applied Biosystems TaqMan® MicroRNA Assays. Applied Biosystems recommend seeding each reverse transcription with 1-10 ng of total RNA per 15 ul reaction. I have always used 10 ng of total RNA in the past and this has posed no problems for me. I dilute the resultant cDNA 1:10 for use in quantitative RT-PCR reactions and this gives optimal C_T values of between 25-30.

The first step of the protocol involves preparing an RT master mix. Each RT requires the following components and volumes, which can be scaled up to suit the required number of reverse transcriptions: 0.15 ul dNTPs, 1 ul reverse transcriptase, 1.5 ul RT buffer, 0.19 ul RNase inhibitor and 4.16 ul nuclease-free water. The master mix should then be gently mixed, centrifuged to bring to the bottom of the tube and then placed on ice. Each RT reaction should then be prepared at a ratio of 7 ul RT master mix:5 ul total RNA. In the past, I have diluted my RNA samples to 2 ng/ul and then seeded each RT with 5 ul of this diluted sample. After mixing, 12 ul of this mix should be added to a 0.2 mL polypropylene reaction tube, to which 3 ul of RT primer should be added. If a large number of RT reactions are being carried out, it is possible to use 96-well reaction plates. The final mix then needs to be centrifuged and then incubated on ice for 5 minutes. Finally, the mix is subjected to the following program of heating: 30 min at 16°C, 30 min at 42°C, 5 min at 85°C and hold at 4°C.

I have used the TaqMan® MicroRNA Reverse Transcription Kit frequently over the past 6 months and have always experienced high quality, reliable cDNA that poses no problems for microRNA detection in quantitative RT-PCR. The kit is easy to use and the RT process is rapid, taking approximately 80 minutes from total RNA to cDNA, including preparation of the RT master mix and heating time. The major advantage of the kit is that very little total RNA is required, as little as 1 ng. This is particularly advantageous when samples are limiting. The disadvantage is that the kit is relatively expensive and the use of microRNA-specific RT primers means the user is limited to pre-defined sequences. This can also be disadvantageous if the user is interested in both the pri-microRNA and mature microRNA sequences, as primers target only the mature. Overall, however, the kits are reliable, rapid to use and capable of producing cDNA from a very small amount of seed RNA.