MicroRNAs are small endogenous RNAs that play a crucial role in translational repression of mRNA transcripts. Over the past decade, miRNA studies have gained high significance due to the important role they play in cellular processes. miRNA are approximately 22 nucleotides long. Their extremely small size makes them difficult to reverse transcribe efficiently. Life Technologies has come up with the TaqMan® MicroRNA Reverse Transcription Kit to address this issue.
The kit has several advantages over conventional reverse transcription kits. First, in this method, cDNA is reverse transcribed from total RNA using miRNA-specific primers for each miRNA to be studied. Therefore, using this method, the resultant cDNA is specific to just the miRNA to be studied and not the entire genome. Further, the primers provided are looped to 3’ end and this extends the tail of the cDNA to increase the size. Second, the primers are such that only the mature miRNAs are detected and not the precursors. The assay is very sensitive and can be used with very small quantities of total RNA.
The kit comes with all the necessary ingredients, including 100mM dNTPs, reverse transcriptase, buffer, RNAse inhibitor and water. The RT primers and PCR primers need to be purchased separately for each miRNA to be studied. The master mix needs to be prepared for each reaction as per the protocol and after addition of total RNA (as low as 10ng), the reaction is performed at 42°C. The entire reaction can be completed within an hour.
We have used the TaqMan MicroRNA Reverse Transcription Kit in our laboratory for the past 8 months and have had excellent results. We were able to reverse transcribe miRNA from cancer cell lines, such as neuroblastoma cell line SK-N-SH. We have used this kit primarily to validate results obtained from a microarray experiments. Even though the techniques are vastly different, using the TaqMan MicroRNA Reverse Transcription Kit, we confirmed alterations of about 15 different miRNAs.
The kit is particularly attractive, due to the sensitivity of the primers. Since each RT primer is specific to its miRNA, false positives are rare. However, this introduces two problems. The most challenging being the introduction of endogenous controls. We cannot have an internal control to account for pipeting errors since control miRNA will need its own primers and cDNA synthesis. To overcome this problem, we tried to use two sets of RT primers, one for test miRNA and the other for internal control miRNA like RNU44, combined in one cDNA synthesis tube. We then used the resultant cDNA for PCR of both RNU44 and the test miRNA. We were able to be successful with this method, but care needs to be taken to see that the two sets of primers don’t cross react.
In summary, the TaqMan MicroRNA Reverse Transcription Kit is a very easy and convenient kit for miRNA studies.