Densitometric gel-like image (virtual gel image) of RNA samples extracted from the same muscle sample using different methods.

<p>The images were generated by a Bio-Rad Experion microfluidic gel electrophoresis system. The first lane is the molecular size marker (ladder), which indicates the approximate size of molecules on the gel. For RNA samples extracted using TRIzol (Lane 2) or an extraction kit containing a Tri-reagent lysis step and precipitating RNA using 2-propanol (Lane 3), 28S and 18S rRNA are clearly visible as two sharp bands. For RNA samples extracted using an extraction kit containing a Tri-reagent lysis step and precipitating RNA using ethanol (Lane 4), 28S and 18S rRNA appear as a smear (indicating the accumulation of low molecular weight components) rather than sharp bands. The location of 28S and 18S rRNA is indicated beside the gel image.</p

Function of common reference genes used in exercise studies.

<p>Function of common reference genes used in exercise studies.</p

Evaluation of reference genes using RefFinder.

<p>Evaluation of reference genes using RefFinder.</p

The sequential stages of the quantitative real-time PCR workflow.

<p>Skeletal muscle samples are cleaned quickly and snap-frozen in liquid nitrogen after the muscle biopsy, and then stored at -80°C until RNA extraction. RNA is extracted using TRIzol or another Tri-reagent, and assessed for concentration and quality. RNA that passes the quality test is used to synthesise cDNA, which is used as template in the final qPCR assay. The conditions of the final qPCR assay must be optimised for each experiment, and the data should be processed using the correct analysis methods.</p

RNA concentration, yield, and quality with different RNA extraction protocols.

<p>RNA concentration, yield, and quality with different RNA extraction protocols.</p

An example of an amplification efficiency test using <i>Cyclophilin</i> (see Table 4 for details) and <i>PPARG coactivator 1 alpha</i> (<i>PGC-1α</i>, see Experiment 4 for details) primers.

<p>A standard curve was generated using a 10-fold dilution of cDNA as template for qPCR reactions. The resulting C_q values are plotted against the Log of the cDNA input. The efficiency, as well as the R² value, are within the acceptable range. The efficiencies of <i>Cyclophilin</i> and <i>PGC-1α</i> are approximately equal, as the absolute value of the slope of ΔC_q against the Log of the cDNA input is < 0.1.</p

An example of the melting curve analysis of a single RNA sample under three different storage/treatment conditions.

<p>The qPCR reaction using cDNA synthesised from Intact (pink) and Degraded RNA (red) sample show the same melting curve, indicating that the same PCR amplicon is produced. However, a different melting curve is observed when using cDNA synthesised from RNase Treated RNA sample (blue), which shows a different PCR amplicon is produced during the qPCR reaction. Our recommendations for qPCR optimisation are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196438#pone.0196438.box006" target="_blank">Box 6</a>.</p

Expression of <i>PGC-1α</i> mRNA in an exercise study with 9 participants.

<p>Muscle samples were taken at rest (Baseline, Week 0) and immediately post-exercise (0 h), and 3 h post-exercise. Data were analysed using 3 different normalisation methods. Values are fold change ± SD.</p

RNA concentration, yield, and quality with different sample handling procedures.

<p>RNA concentration, yield, and quality with different sample handling procedures.</p

Example of a primer specificity test.

<p>DNA fragment products produced from a PCR reaction with the same primers, but using either water (Lane 2), -RT (cDNA synthesis reaction containing RNA but no cDNA, Lane 3), or cDNA (Lane 4) as template, were separated on a 2% agarose gel. Lane 1 is the 100 bp DNA ladder. A single sharp DNA band of the expected size, which is the final PCR amplicons, is present only in the reaction with cDNA.</p