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Gene expression studies require high quality cDNA. The TaqMan® MicroRNA
Reverse Transcription Kit delivers a high level of performance
for accuracy, precision, and quality of cDNA. The kit provides
the necessary components for optimal performance of TaqMan
MicroRNA Assays (Figure 3). Components of this kit are used with the Reverse
Transcription (RT) primer provided with the specific TaqMan
MicroRNA Assay to convert miRNA to cDNA.
Case Study In our study, an RNA mass equivalent of 5.13 μg of
tissue was added to a final RT reaction volume of 15 μL.
RT was performed in 384 well format using Applied Biosystems
TaqMan MicroRNA Reverse Transcription Kit Protocol.
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| Figure 3. TaqMan® MicroRNA Assay Mechanism. (A) A simple, two-step mechanism brings the advantages of real-time PCR to miRNA quantitation. The basis of TaqMan MicroRNA Assays is a target-specific stem-loop structure, reverse-transcription primer. Its innovative design overcomes a fundamental problem in miRNA quantification: the short length of mature miRNAs (~22 nt) prohibits conventional design of a random-primed RT step followed by a specific real-time assay. (B) The stem-loop accomplishes two goals: (1) specificity for only mature miRNA target, and (2) formation of a RT primer/mature miRNA–chimera, extending the 5' end of the miRNA. The resulting longer RT amplicon presents a template amenable to standard real-time PCR, using TaqMan assays. |
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