Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs.
Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.
Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney.
Wilson
W
Wong
Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney.
Ryan
R
Farr
Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney.
Mugdha
M
Joglekar
Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney.
Andrzej
A
Januszewski
Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney; anand.hardikar@ctc.usyd.edu.au.
Anandwardhan
A
Hardikar
D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D044085:Microfluidics / Q000379:methods; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
D018076:DNA, Complementary; D035683:MicroRNAs