qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data [Full article]
The qBase paper describes the mathematical framework and quantification principles that are implemented in Biogazelle's qBasePlus software. It also discusses some important issues of experiment design (sample versus gene maximization strategy) that affect the way in which a real-time PCR experiment can or must be analyzed.
RTPrimerDB: The Real-Time PCR Primer and Probe Database [Full article]
The real-time polymerase chain reaction (PCR) methodology has become increasingly popular for nucleic acids detection and/or quantification. As primer/probe design and experimental evaluation is time-consuming, we developed a public database application for the storage and retrieval of validated real-time PCR primer and probe sequence records. The integrity and accuracy of the data are maintained by linking to and querying other reference databases. RTPrimerDB provides free public access through the Web to perform queries and submit user based information. Primer/probe records can be searched for by official gene symbol, nucleotide sequence, type of application, detection chemistry, LocusLink or Single Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve additional information on the gene/SNP for which the primers/probes are designed. Currently, the database contains primer/probe records for human, mouse, rat, fruit fly and zebrafish, and all current detection chemistries such as intercalating dyes (SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons.
RTPrimerDB: the real-time PCR primer and probe database, major update 2006 [Full article]
This second paper on the Real-time PCR Primer and Probe Database (RTPrimerDB) reports on the free in silico PCR assay evaluation pipeline for automated prediction of qPCR assay performance.
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [Full article]
Vandesompele et al. (2002) were the first to quantify the errors associated with the use of a single (non-validated) reference gene, to develop a method to select the most stably expressed reference genes, and to propose the use of multiple reference genes for calculation of a reliable normalization factor.
A new mathematical model for relative quantification in real-time RT-PCR [Full article]
Modification of the original delta-delta-Ct quantification model through correction of PCR efficiency, while still using only one reference gene for normalization.
Analysis of relative gene expression data using real-time quantitative PCR and the 2^(-delta-delta-Ct) Method [Full article]
Original publication of the delta-delta-Ct relative quantification model in which PCR efficiency is assumed to be 100% and normalization is performed using one reference gene.
Quantification of splice variants using real-time PCR [Full article]
A reliable and robust method for measuring the expression of alternatively spliced transcripts is an important step in investigating the significance of each variant. So far, accurate quantification of splice variants has been laborious and difficult due to the intrinsic limitations of conventional methods. The many advantages of real-time PCR have made this technique attractive to study its application in quantification of splice isoforms. We use skipping of exon 37 in the NF1 gene as a model to compare and evaluate the different strategies for quantitating splice variants using real-time PCR. An overview of three different possibilities for detecting alternative transcripts is given. We propose the use of a boundary-spanning primer to quantify isoforms that differ greatly in abundance. We describe here a novel method for creating a reliable standard curve using one plasmid containing both alternative transcripts. In addition, we validate the use of an absolute standard curve based on a dilution series of fluorometrically quantified PCR products.
Elimination of primer-dimer artefacts and genomic co-amplification using a two-step SYBR Green I real-time RT-PCR [Full article]
Rapid detection of VHL exon deletions using real-time quantitative PCR [Full article]
Various types of mutations exist that exert an effect on the normal function of a gene. Among these, exon/gene deletions often remain unnoticed in initial mutation screening. Until recently, no fast and efficient methods were available to detect this type of mutation. Molecular detection methods for gene copy number changes included Southern blot (SB) and fluorescence in situ hybridisation, both with their own intrinsic limitations. In this paper, we report the development and application of a fast, sensitive and high-resolution method for the detection of single exon or larger deletions in the VHL gene based on real-time quantitative PCR (Q-PCR). These deletions account for approximately one-fifth of all patients with the von Hippel-Lindau syndrome, a dominantly inherited highly penetrant familial cancer syndrome predisposing to specific malignancies including phaeochromocytomas and haemangioblastomas. Our VHL exon quantification strategy is based on SYBR Green I detection and normalisation using two reference genes with a normal copy number, that is, ZNF80 (3q13.31) and GPR15 (3q12.1). Choice of primer sequences and the use of two reference genes appears to be critical for accurate discrimination between 1 and 2 exon copies. In a blind Q-PCR study of 29 samples, all 14 deletions were detected, which is in perfect agreement with previously determined SB results. We propose Q-PCR as the method of choice for fast (within 3.5 h), accurate and sensitive (ng amount of input DNA) exon deletion screening in routine DNA diagnosis of VHL disease. Similar assays can be designed for deletion screening in other genetic disorders.
Impact of RNA quality on reference gene expression stability [Full article]
Real-time quantitative allele discrimination assay using 3" LNA primers for detection of low-percentage mosaic mutations [Full article]
Standardization of real-time PCR gene expression data from independent biological replicates [Full article]
Gene expression analysis by quantitative reverse transcription PCR (qRT-PCR) allows accurate quantifications of messenger RNA (mRNA) levels over different samples. Corrective methods for different steps in the qRT-PCR reaction have been reported; however, statistical analysis and presentation of substantially variable biological repeats present problems and are often not meaningful, for example, in a biological system such as mouse embryonic stem cell differentiation. Based on a series of sequential corrections, including log transformation, mean centering, and autoscaling, we describe a robust and powerful standardization method that can be used on highly variable data sets to draw statistically reliable conclusions.