title: No control genes required: Bayesian analysis of qRT-PCR data.
abstract: BACKGROUND
  Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR) is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process.
  
  
  RESULTS
  In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC) algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts). Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization) but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests.
  
  
  CONCLUSIONS
  Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been implemented as the MCMC.qpcr package in R.
authors: Department of Integrative Biology, University of Texas at Austin, Austin, Texas, United States of America.
authors: Mikhail V
authors: MV
authors: Matz
authors: Rachel M
authors: RM
authors: Wright
authors: James G
authors: JG
authors: Scott
mesh_terms: D000818:Animals; D037421:Anthozoa / Q000235:genetics; D001499:Bayes Theorem; D030541:Databases, Genetic; D005796:Genes / Q000235:genetics*; D016014:Linear Models; D008390:Markov Chains; D008957:Models, Genetic; D009010:Monte Carlo Method; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013312:Stress, Physiological / Q000235:genetics
references: Methods Mol Biol. 1998;110:43-61
references: 9918038
references: Genomics. 2009 Aug;94(2):146-52
references: 19422910
references: Biotechniques. 2001 Sep;31(3):502, 504-6, 508
references: 11570493
references: Mar Biotechnol (NY). 2010 Oct;12(5):594-604
references: 20041338
references: Genome Biol. 2007;8(2):R19
references: 17291332
references: Biometrics. 1987 Sep;43(3):671-81
references: 3663823
references: Nat Rev Genet. 2006 Jan;7(1):55-65
references: 16369572
references: Lasers Surg Med. 2010 Dec;42(10):752-65
references: 21246580
references: Nucleic Acids Res. 2001 May 1;29(9):e45
references: 11328886
references: BMC Bioinformatics. 2008 Apr 29;9:221
references: 18445269
references: Genome Res. 1996 Oct;6(10):986-94
references: 8908518
references: Methods. 2001 Dec;25(4):402-8
references: 11846609
references: J Biotechnol. 1999 Oct 8;75(2-3):291-5
references: 10617337
references: Ann Oncol. 1997 Jul;8(7):633-6
references: 9296214
references: Nucleic Acids Res. 2003 Oct 15;31(20):e122
references: 14530455
references: Anal Biochem. 2002 Mar 1;302(1):52-9
references: 11846375
references: Nucleic Acids Res. 1996 Jun 1;24(11):2194-5
references: 8668555
references: Biotechniques. 2005 Feb;38(2):287-93
references: 15727135
references: J Mol Endocrinol. 2000 Oct;25(2):169-93
references: 11013345
references: PLoS One. 2012;7(1):e29788
references: 22238653
references: Cancer Res. 2004 Aug 1;64(15):5245-50
references: 15289330
references: Brain Res. 2011 Mar 24;1381:11-20
references: 21094150
references: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references: 12184808
references: J Stat Softw. 2010;33(1):1-22
references: 20808728
references: PLoS One. 2011;6(10):e26914
references: 22046408
references: Parasitology. 2001 May;122(Pt 5):563-9
references: 11393830
references: BMC Bioinformatics. 2006 Feb 22;7:85
references: 16504059
references: Brain Res. 2006 Jan 5;1067(1):36-42
references: 16271354
references: Anal Biochem. 2000 Feb 15;278(2):175-84
references: 10660460
references: Nucleic Acids Res. 2006;34(18):e123
references: 17000636
references: BMC Mol Biol. 2010 Aug 11;11:57
references: 20701777