Fig 1.
A. Summary of the key steps leading from gene to protein expression in eukaryotes. DNA is first transcribed into RNA, then processed to mRNA after removing the noncoding regions (introns, green) and splicing the coding regions (exons, red) together. The spliced mRNA (red) is then exported to the cytoplasm to produce the protein molecule. B. Outline of procedures for first strand complementary DNA (cDNA) synthesis from messenger RNA (mRNA) using short sequences of deoxy-thymidine nucleotides (oligo-dT primers, green). After annealing of Oligo-dT primers to the mRNA sequence (grey), the RNA-directed DNA polymerase, reverse transcriptase, is able to synthesise cDNA strand (blue), as well as degrade mRNA (red) from the hybrid molecule.
Fig 2.
The sequential stages of the quantitative real-time PCR workflow.
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.
Fig 3.
Densitometric gel-like image (virtual gel image) of RNA samples extracted from the same muscle sample using different methods.
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.
Table 1.
RNA concentration, yield, and quality with different sample handling procedures.
Table 2.
RNA concentration, yield, and quality with different RNA extraction protocols.
Table 3.
RNA concentration and quality of different storage methods.
Fig 4.
Example of a primer specificity test.
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.
Fig 5.
An example of the melting curve analysis of a single RNA sample under three different storage/treatment conditions.
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 Box 6.
Fig 6.
An example of an amplification efficiency test using Cyclophilin (see Table 4 for details) and PPARG coactivator 1 alpha (PGC-1α, see Experiment 4 for details) primers.
A standard curve was generated using a 10-fold dilution of cDNA as template for qPCR reactions. The resulting Cq 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 Cyclophilin and PGC-1α are approximately equal, as the absolute value of the slope of ΔCq against the Log of the cDNA input is < 0.1.
Table 4.
Function of common reference genes used in exercise studies.
Fig 7.
Expression of six commonly-used reference genes in exercise studies.
Cq values of individual reactions using ACTB, TBP, Cyclophilin, GAPDH, B2M, and 18S rRNA primers are presented. All samples are from an exercise study (n = 9 participants × 3 time points = 27 samples).
Table 5.
Evaluation of reference genes using RefFinder.
Table 6.
Primer sequences and amplicon details.
Fig 8.
A: Determination of cDNA amount in reactions with different RNA input. Different amounts of RNA were used to synthesise cDNA (n = 4 for each RNA input), and the relative amount of cDNA in each reaction was measured using OliGreen dye. Values are presented as mean ± SD. B: Correlation between RNA input and average relative amount of cDNA measured.
Fig 9.
Expression of PGC-1α mRNA in an exercise study with 9 participants.
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.