Digitial PCR Applications: Gene Expression Analysis

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Gene expression comprises multiple cellular processes, from transcription, processing, and export of mRNA from the nucleus to translation into protein and post-translational modifications. Each stage is tightly regulated by the cell, and overall expression levels reflect the sum of these steps. Molecular techniques are commonly used for assessing gene expression because they are fast, sensitive, and relatively inexpensive. mRNA quantification exploits antisense hybridization either directly, as in the case of microarray, RNase protection assay, and northern blotting, or with subsequent amplification, such as polymerase chain reaction (PCR).

Digital PCR (dPCR) is a valuable tool to complement other molecular techniques across a wide range of applications, including gene expression analysis. dPCR divides the reaction into many smaller micro-reactions, each of which is read individually and interpreted as positive or negative based on the presence or absence of fluorescence (endpoint detection).

Separating the targets into micro-reactions permits direct quantification of the target sequence (through Poisson statistics) without the need for reference standards or a standard curve. Digital PCR excels at providing highly precise quantification, even at low concentrations, and can often detect small differences more reliably than other techniques. Because it relies on endpoint detection, dPCR results are not impacted by variations in PCR efficiency and is generally more robust and reliable for working with challenging samples that contain PCR inhibitors or low-abundance targets.

Explore more details about gene expression analysis by dPCR in our technical note.

Frequently asked questions

dPCR can provide absolute quantification of target mRNAs without a standard curve. The high accuracy, precision, and sensitivity of dPCR enables reliable quantification of low-abundance targets and detection of small changes in target abundance.

dPCR complements qPCR in gene expression studies. Whereas the wide dynamic range and throughput of qPCR is ideal for routine gene expression analysis, dPCR excels at quantifying low-abundance targets and small changes. Furthermore, dPCR is more tolerant of assays with varying efficiencies and inter-assay competition, which provides greater flexibility for multiplexing.

Because the reaction is compartmentalized, dPCR is more tolerant of PCR inhibitors than qPCR. Nucleic acid extraction techniques vary in their ability to remove common PCR inhibitors that are often present in biological and environmental samples. Some inhibitors co-purify with nucleic acids and can be difficult or impossible to remove completely. Whether your sample contains inhibitors or is limited, the high sensitivity and high precision of dPCR can enable more robust quantification of gene expression than other molecular techniques.