Validation of a multiplex reverse transcription and pre-amplification method using TaqMan(®) MicroRNA assays

Joane Le Carré, Séverine Lamon, Bertrand Léger, Joane Le Carré, Séverine Lamon, Bertrand Léger

Abstract

Since the discovery of microRNAs (miRNAs), different approaches have been developed to label, amplify and quantify miRNAs. The TaqMan(®) technology, provided by Applied Biosystems (ABIs), uses a stem-loop reverse transcription primer system to reverse transcribe the RNA and amplify the cDNA. This method is widely used to identify global differences between the expression of 100s of miRNAs across comparative samples. This technique also allows the quantification of the expression of targeted miRNAs to validate observations determined by whole-genome screening or to analyze few specific miRNAs on a large number of samples. Here, we describe the validation of a method published by ABIs on their web site allowing to reverse transcribe and pre-amplify multiple miRNAs and snoRNAs simultaneously. The validation of this protocol was performed on human muscle and plasma samples. Fast and cost efficient, this method achieves an easy and convenient way to screen a relatively large number of miRNAs in parallel.

Keywords: human skeletal muscle; miRNA; multiplexing; plasma; qRT-PCR.

Figures

FIGURE 1
FIGURE 1
Method design.
FIGURE 2
FIGURE 2
(A) Standard curves from 11 miRNAs expressed in human muscle tissue. (B) Standard curves from eight microRNAs (miRNAs) expressed in human plasma samples.

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