Measuring gene expression by RT-qPCR

A gene is switched on when it is copied into messenger RNA (mRNA). Measuring how much mRNA of a gene a tissue contains is therefore a way to ask whether a treatment or a disease changes the activity of that gene. RT-qPCR does this in two steps. Reverse transcription (RT) copies the RNA into complementary DNA (cDNA), which is stable; quantitative PCR (qPCR) then amplifies a short stretch of the cDNA of the chosen gene while a fluorescent dye reports, cycle by cycle, how much double-stranded DNA has been made.

The fewer cycles a reaction needs to produce a detectable amount of fluorescence, the more copies of the target it started with; Higuchi and colleagues introduced this kinetic, real-time way of reading PCR (Higuchi et al., 1993). The cycle at which a reaction crosses a fluorescence threshold is called the quantification cycle, Cq. At 100% efficiency the product doubles every cycle, so a difference of one cycle means a twofold difference in starting template. With a dye such as SYBR Green, which binds any double-stranded DNA, heating the products at the end of the run gives a melt curve that tells the intended product apart from unwanted ones (Ririe et al., 1997).

Most of the work happens before the PCR. RNA is easily degraded, so the bench must be kept free of ribonucleases (RNases). RNA is commonly extracted with an acid guanidinium thiocyanate–phenol–chloroform mixture (Chomczynski and Sacchi, 1987) or with silica columns; its purity is checked by absorbance and its integrity by capillary electrophoresis, which gives an RNA integrity number (RIN) between 1 and 10 (Schroeder et al., 2006). Integrity affects how well RT-qPCR performs (Fleige and Pfaffl, 2006). Each sample is then compared with reference genes, whose expression should not differ between the groups; several carefully chosen reference genes are more reliable than one (Vandesompele et al., 2002).

The target is Gx, a hypothetical gene, measured in the hippocampus of mice treated with a drug or vehicle, or of human donors with Alzheimer's disease and control donors from a brain bank. The simulator records the true expression of Gx, so the decisions that changed the result are shown at the end. Relative quantification follows the 2^−ΔΔCq method (Livak and Schmittgen, 2001) and the efficiency-corrected method of Pfaffl (Pfaffl, 2001), and the report follows the MIQE guidelines (Bustin et al., 2009).

Learning objectives

• Design an expression study with the animal or donor as the unit of analysis, random allocation or matched donors, and coded samples
• Work RNase-free, extract RNA with phenol–chloroform or a silica column, and judge quantity, purity (A260/280, A260/230) and integrity (RIN)
• Explain why DNase treatment, no-RT controls and primers that span an exon–exon junction protect against amplifying genomic DNA
• Calculate amplification efficiency from the slope of a standard curve, and read amplification curves, the threshold, Cq values and melt curves, including primer dimers
• Choose and validate reference genes, and calculate relative expression by the ΔΔCq and the efficiency-corrected (Pfaffl) methods
• Test ΔCq values with one value per animal or donor, report a fold change with its confidence interval, and check the report against MIQE

Ethics and safety

Mouse tissue comes from animals used under a protocol approved by an Institutional Animal Ethics Committee (in India, the CCSEA under the Prevention of Cruelty to Animals Act 1960; elsewhere, frameworks such as EU Directive 2010/63/EU, the US Animal Welfare Act and PHS Policy, and the UK Animals (Scientific Procedures) Act 1986); group sizes are justified in advance, tissue from one set of animals can serve several assays (Reduction, one of the 3Rs), and the work is reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020). Human brain tissue comes only from people who consented to donation, usually through a brain bank, and its use requires approval by an ethics committee (in India, an Institutional Ethics Committee following the ICMR National Ethical Guidelines of 2017; elsewhere, a research ethics committee or, in the United States, an institutional review board); donors are compared by diagnosis, nothing is done to them, and donor details are kept confidential. Post-mortem RNA quality depends mostly on the circumstances before death, such as coma, hypoxia and ventilation, with brain pH a useful indicator (Tomita et al., 2004; Durrenberger et al., 2010). Fresh-frozen tissue is used for RNA, because formalin fixation cross-links and fragments it; lipofuscin autofluorescence, a problem for imaging aged human tissue, does not affect qPCR, which measures extracted nucleic acid in solution. At the bench, phenol–guanidine reagents and chloroform are toxic and are handled in a fume hood with gloves and eye protection; dry ice needs insulated gloves and a ventilated room.

Key terms

• RNase: An enzyme that cuts RNA. RNases are present on skin, in dust and in tissue itself, and some are very stable, so RNA work needs clean surfaces and RNase-free tips and tubes.
• RT-qPCR: Reverse transcription followed by quantitative PCR: RNA is copied into cDNA, and the cDNA of one gene is amplified while fluorescence is measured in every cycle.
• cDNA: Complementary DNA: a DNA copy of RNA made by the enzyme reverse transcriptase.
• A260/280 and A260/230: Ratios of absorbance at 260 nm (nucleic acid) to 280 nm (protein, phenol) and to 230 nm (guanidine salts, phenol). Pure RNA gives approximately 2.0 for both.
• RIN: RNA integrity number, from 1 (degraded) to 10 (intact), calculated from the electropherogram of the RNA, in which intact RNA shows two sharp ribosomal peaks (18S and 28S).
• DNase I: An enzyme that digests DNA, used to remove genomic DNA from an RNA preparation before reverse transcription.
• No-RT control: The RNA of a sample taken through reverse transcription without the enzyme. Any signal it gives comes from DNA, not from RNA.
• Exon–exon junction: The point in an mRNA where two exons meet after splicing. A primer that spans it cannot bind genomic DNA, where an intron lies between the exons.
• Cq: Quantification cycle: the (fractional) cycle at which the fluorescence of a reaction crosses the threshold. A lower Cq means more starting template.
• Amplification efficiency: The fraction of templates copied in each cycle; 100% means exact doubling. Calculated from the slope of a standard curve: efficiency = 10^(−1/slope) − 1.
• Melt curve: Fluorescence measured while the products are heated; each double-stranded product separates at its own melting temperature, giving a peak in −dF/dT.
• Primer dimer: A short product made when two primers bind to each other and are extended; it melts at a lower temperature than the real product.
• Reference gene: A gene whose expression is assumed not to differ between the groups, used to correct for differences in RNA input and reverse transcription between samples.
• ΔCq and ΔΔCq: ΔCq = Cq of the target minus Cq of the reference genes for one sample; ΔΔCq = that sample's ΔCq minus the mean ΔCq of the control group. Fold change = 2^−ΔΔCq at 100% efficiency.