Document Type

Article

Publication Date

2009

Journal or Book Title

Current Issues in Molecular Biology

Volume

12

Issue

3

First Page

129

Last Page

134

Abstract

Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses. To address this, we invented the PREXCEL-Q software program to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with ~15,500 SPUD amplicons yielded a Cq of 27.39 +/- 0.28 (at ~80.8% efficiency), while reactions spiked with ~7,750 SPUD plasmids yielded a Cq of 23.82 +/- 0.15 (at ~97.85% efficiency). This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.

Comments

This article is from Current Issues in Molecular Biology 12, no. 3 (2009): 129–134.

Copyright Owner

The authors

Language

en

File Format

application/pdf

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