PCR is a popular technique for mRNA detection, measurement, and validation in gene expression studies. In the last decade, qPCR methods have been developed to measure gene expression in studies ranging from DNA methylation to microRNA (miRNA) detection.
In Bio-Rad's Droplet Digital™ PCR (ddPCR™) System, each sample is partitioned into many droplets prior to amplification. After cycling, the level of fluorescence is measured in each droplet; droplets with fluorescence are scored as positive, and those with little or no fluorescence are scored as negative. Application of Poisson statistical analysis yields absolute quantification. ddPCR technology does not rely on a standard curve and is insensitive to the number of amplification cycles. One of the greatest advantages of digital PCR for gene expression studies is the exquisite sensitivity of quantification it provides. This sensitivity is particularly evident in quantification of low-abundance target, and in discrimination of rare alleles against an abundant wild-type background.
Regulation of gene expression at the genomic level is primarily controlled by the methylation of genomic DNA and by DNA-histone interactions, which are affected by histone modifications such as methylation, phosphorylation, and acetylation. Genomic DNA methylation usually occurs at CpG islands in noncoding regions and generally reduces gene expression.
PCR is one of several techniques used for studying methylation. The most common PCR-based method for the quantification of DNA methylation is the use of different primer pairs that are designed to distinguish methylated from unmethylated CpG islands after treatment with sodium bisulfite (Ku et al. 2011). Unmethylated CpG is converted to UpG by sodium bisulfite, whereas methylated DNA is unaffected. Due to its great capacity to discriminate alleles, digital PCR can provide more sensitive detection and accurate measurement of methylation events. Its extreme precision also makes it a method of choice when operating with low amounts of starting material (such as chromatin immunoprecipitation experiments).
Most analysis of gene expression is focused on quantifying transcription levels. Determination of the presence or absence of particular transcripts and their quantification by reverse-transcription PCR (RT-PCR) is now routine in many laboratories. Digital PCR can increase the detection of rare transcripts and provide easier and more accurate quantitation. Either one-step or two-step RT-PCR can be used in ddPCR.
Detection of rare transcripts — a problem with RT-PCR for the detection of rare transcripts is that in standard PCR there is a bias toward underrepresentation of low-abundance sequences. The partitioning of a PCR reaction into droplets helps reduce this bias. Within each droplet, a rare sequence faces reduced competition from both high-abundance sequences and sequences that are easier to amplify, thus increasing the relative amplification of the target sequence and providing more accurate quantitation.
Measurement of miRNAs — the discovery of miRNAs added another level of complexity to studies of gene expression. Changes in miRNA levels are thought to be involved in all aspects of gene expression regulation, and associated with normal developmental processes as well as diseases ranging from cancer to diabetes. Levels of miRNAs vary over time, and some mature miRNAs display very rapid turnover, necessitating fast, sensitive, and accurate detection.
Recently, miRNAs have been detected in blood. Circulating miRNAs have been shown to be stable and suitable for use as markers of disease including cancer (Mitchell et al. 2008). The detection of miRNA in blood and other fluids such as saliva has paved the way for the development of sensitive new and complementary methods for the diagnosis and monitoring of many diseases. ddPCR has been demonstrated to be an excellent tool for the study of miRNAs (Hindson et al. 2013)
Digital PCR can be used for studies of gene expression both alone and in combination with other techniques. One advantage of the QX200™ ddPCR System is that it is compatible with existing primers, probes, and protocols for gene expression studies. It even provides the capacity to do simultaneous detection of target and reference using EvaGreen chemistry (McDermott et al. 2013), making the study of gene expression even simpler and more precise.
Hindson CM et al. (2013). Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10, 1003–1005. PMID: 23995387
Ku JL et al. (2011). Methylation-specific PCR. Methods Mol Biol 79, 123–32. PMID: 21913069
McDermott GP et al. (2013). Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR. Anal Chem 8511, 619–11627. PMID: 24180464
Mitchell PS et al. (2008). Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105, 10513–10518. PMID: 18663219