High Throughput Quantitative PCR Using Low-input Samples for mRNA and MicroRNA Gene Expression Analyses
Year: 2013
Type: article
Abstract: Technical advancements in quantitative PCR (qPCR) instrumentation have made it possible to perform gene expression measurements using small sample input to support both basic and clinical research studies. As part of the strategic goals to assess new technologies and identify protocols that best fit the needs of the Mayo Clinic, we compared the Fluidigm BioMark system with standard Applied Biosystems (AB) instrumentation for mRNA and miRNA gene expression measurements. We also examined the performance of the BioMark system when using very low-input RNA. We evaluated a set of control samples using the same TaqMan assays with both systems. We observed that the BioMark-generated data routinely yields Ct values approximately 10 cycles lower than those obtained with AB instrumentation. The correlations between the two platforms were high (r = 0.96) for both mRNA and miRNA expression experiments. For miRNA expression, a similarly high correlation was observed between fresh frozen and formalin-fixed paraffin embedded (FFPE) samples. In an effort to accommodate our customer needs, we also evaluated the performance of the BioMark for evaluating gene expression in very low-input samples. Using six standard TaqMan control assays (having high, medium and low expression levels), we observed that high quality RNA samples as low as 10pg achieved linear amplification across four different pre-amplification cycles (10, 14, 18 and 22). At 10pg total RNA input, low-expression control assay IPO8 demonstrated a correlation of r = .999 among the four pre-amplification cycles. This linearity was also observed at higher RNA input levels, up to 10ng. The only control assay that did not perform in a linear fashion across all input amounts and all pre-amplification cycles was 18S ribosomal RNA. The highest correlation observed for 18S was r = 0.801, and this supports the vendor suggestion that 18S is not the best control assay option.
Source: Europe PMC (PubMed Central)
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W936278666