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Biomol Detect Quantif. 2017 Sep 20;13:40-48. doi: 10.1016/j.bdq.2017.09.001. eCollection 2017 Sep.

Evaluation of relative quantification of alternatively spliced transcripts using droplet digital PCR.

Author information

1
Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium.
2
Department of Basic Medical Sciences, Ghent University, Ghent, Belgium.
3
Cancer Research Institute Ghent (CRIG), Ghent, Belgium.
4
HIV Translational Research unit, Department of Internal Medicine, Ghent University, Ghent, Belgium.
5
Bioinformatics Institute Ghent (BIG), Ghent, Belgium.

Abstract

INTRODUCTION:

For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms.

MATERIALS AND METHODS:

RNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method.

RESULTS:

Both ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments.

CONCLUSIONS:

Our study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.

KEYWORDS:

Alternative splicing; Droplet digital PCR; Reverse transcriptase polymerase chain reaction

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