Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 98, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/52586
Keywords
Molecular Biology; Issue 98; microRNA; ncRNA; probe-based assays; high-throughput PCR; Nanofluidics / Open Arrays; reverse-transcription; pre-amplification; qPCR
Categories
Funding
- Australian Research Council [FT110100254]
- JDRF, Australia [CRN201314]
- Australian Research Council [FT110100254] Funding Source: Australian Research Council
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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.
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