The Applied Biosystems™ TaqMan™ Advanced miRNA cDNA Synthesis Kit uses a universal reverse transcription (RT) chemistry to prepare the cDNA template for use with TaqMan™ Advanced miRNA Assays
for detection and quantification of mature miRNAs in biological samples.
Features of the TaqMan Advanced miRNA workflow include:
high sensitivity allows for quantification of low-expressing miRNA targets, especially in biological fluids such as plasma, serum, and tissue
the universal RT chemistry generates universal cDNA templates for all miRNAs present in the sample, allowing for flexibility in study design
• Sample savings:
requires only 2 μL of plasma/serum sample input and generates enough cDNA for 600 qPCR reactions (20 μL) or 1200 qPCR reactions (10 μL)
5’ ligation step allows for single base pair discrimination along entire miRNA sequence, including difficult-to-detect mismatches on 5’ end
• Gold-standard TaqMan chemistry:
This kit is designed for use with TaqMan® Advanced miRNA Assays for qPCR, which draw on Thermo Fisher Scientific's industry-leading bioinformatics assay design pipeline and validation system to help ensure high specificity and minimal cross-reactivity, even for closely-related miRNA families.
The TaqMan Advanced miRNA cDNA Synthesis Kit uses 3' poly-A tailing and 5' ligation of an adaptor sequence to extend the mature miRNAs present in the sample on each end prior to reverse transcription. Universal RT primers recognize the universal sequences present on both the 5' and 3' extended ends of the mature miRNAs. All mature miRNAs in the sample are reverse transcribed to cDNA.
In order to improve detection of low-expressing miRNA targets, the cDNA is then amplified using the Universal miR-Amp Primers and miR-Amp Master Mix to uniformly increase the amount of cDNA for each target, maintaining the relative differential expression levels. Unlike traditional pre-amplification, the Universal miR-Amp Primers recognize the universal sequences added to all mature miRNAs on the 5' and 3' ends, ensuring that there is no amplification bias.Note:
the TaqMan Advanced miRNA cDNA Synthesis Kit is not compatible with first generation TaqMan MicroRNA Assays for qPCR. For reverse transcription of miRNA targets using miRNA-specific RT primers, please see the TaqMan™ MicroRNA Reverse Transcription Kit