Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
Jo Vandesompele,Katleen De Preter,Filip Pattyn,Bruce Poppe,Nadine Van Roy,Anne De Paepe,Franki Speleman +6 more
Reads0
Chats0
TLDR
The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which opens up the possibility of studying the biological relevance of small expression differences.Abstract:
Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.read more
Citations
More filters
Journal ArticleDOI
Analysis of Immune Signatures in Longitudinal Tumor Samples Yields Insight into Biomarkers of Response and Mechanisms of Resistance to Immune Checkpoint Blockade.
Pei Ling Chen,Whijae Roh,Alexandre Reuben,Zachary A. Cooper,Christine N. Spencer,Peter A. Prieto,John P. Miller,Roland L. Bassett,Vancheswaran Gopalakrishnan,Khalida Wani,Mariana Petaccia de Macedo,Jacob Austin-Breneman,Hong Jiang,Qing Chang,Sangeetha M. Reddy,Wei Shen Chen,Michael T. Tetzlaff,Russell J. Broaddus,Michael A. Davies,Jeffrey E. Gershenwald,Lauren E. Haydu,Alexander J. Lazar,Sapna Pradyuman Patel,Patrick Hwu,Wen-Jen Hwu,Adi Diab,Isabella C. Glitza,Scott E. Woodman,Luis M Vence,Ignacio I. Wistuba,Rodabe N. Amaria,Lawrence N. Kwong,Victor G. Prieto,R. Eric Davis,Wencai Ma,Willem W. Overwijk,Arlene H. Sharpe,Jianhua Hu,P. Andrew Futreal,Jorge Blando,Padmanee Sharma,James P. Allison,Lynda Chin,Jennifer A. Wargo +43 more
TL;DR: It is demonstrated that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade.
Journal ArticleDOI
Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage
Wong Connie C,Kevin E. Loewke,Nancy L. Bossert,Barry Behr,Christopher J. De Jonge,Thomas M. Baer,Renee A. Reijo Pera +6 more
TL;DR: It is found that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA).
Journal ArticleDOI
Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process
Marino Exposito-Rodriguez,Marino Exposito-Rodriguez,Andrés A. Borges,Andrés Borges-Pérez,José A. Pérez +4 more
TL;DR: This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process and suggests that SGN-U314153 (CAC), Sgn-U321250 (TIP41), S GN-U346908 ("Expressed") and SGN -U316474 (SAND) genes provide superior transcript normalization in tomato development studies.
Journal ArticleDOI
Evidence based selection of housekeeping genes
Hendrik J. M. de Jonge,Rudolf S N Fehrmann,Eveline S. J. M. de Bont,Robert M. W. Hofstra,Frans Gerbens,Willem A. Kamps,Elisabeth G.E. de Vries,Ate G.J. van der Zee,Gerard J. te Meerman,Arja ter Elst +9 more
TL;DR: Novel candidate housekeeping genes (e.g. RPS13, RPL27, RPS20 and OAZ1) with enhanced stability among a multitude of different cell types and varying experimental conditions are shown.
Journal ArticleDOI
iRegulon: from a gene list to a gene regulatory network using large motif and track collections.
Rekin's Janky,Annelien Verfaillie,Hana Imrichova,Bram Van de Sande,Laura Standaert,Valerie Christiaens,Gert Hulselmans,Koen Herten,Marina Naval Sanchez,Delphine Potier,Dmitry Svetlichnyy,Zeynep Kalender Atak,Mark Fiers,Jean-Christophe Marine,Stein Aerts +14 more
TL;DR: Over-activate p53 in breast cancer cells, followed by RNA-seq and ChIP-seq, and could identify an extensive up-regulated network controlled directly by p53 and a repressive network with no indication of direct p53 regulation but rather an indirect effect via E2F and NFY are mapped.
References
More filters
Journal ArticleDOI
Quantitative monitoring of gene expression patterns with a complementary DNA microarray.
TL;DR: A high-capacity system was developed to monitor the expression of many genes in parallel by means of simultaneous, two-color fluorescence hybridization, which enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA.
Journal ArticleDOI
Real time quantitative PCR.
TL;DR: Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays.
Journal ArticleDOI
Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.
TL;DR: The technical aspects involved are discussed, conventional and kinetic RT-PCR methods for quantitating gene expression are contrasted, and the usefulness of these assays are illustrated by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
Journal ArticleDOI
Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions
TL;DR: Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range.
Journal ArticleDOI
Systematic variation in gene expression patterns in human cancer cell lines.
Douglas T. Ross,Uwe Scherf,Michael B. Eisen,Charles M. Perou,Christian A. Rees,Paul T. Spellman,Vishwanath R. Iyer,Stefanie S. Jeffrey,Matt van de Rijn,Mark Waltham,Alexander Pergamenschikov,Jeffrey C. Lee,Deval A. Lashkari,Dari Shalon,Timothy G. Myers,John N. Weinstein,David Botstein,Patrick O. Brown +17 more
TL;DR: Using cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.