The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model.
The interaction of Leishmania with BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver…) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation. The development of technical platforms for high-throughput qPCR analysis, and powerful software for analysis of qPCR data, have acknowledged the problem that some reference genes widely used due to their known or suspected "housekeeping" roles, should be avoided due to high expression variability across different tissues or experimental conditions. In this paper we evaluated the stability of 112 genes using three different algorithms: geNorm, NormFinder and RefFinder in spleen samples from BALB/c mice under different experimental conditions (control and Leishmania infantum-infected mice). Despite minor discrepancies in the stability ranking shown by the three methods, most genes show very similar performance as RG (either good or poor) across this massive data set. Our results show that some of the genes traditionally used as RG in this model (i.e. B2m, Polr2a and Tbp) are clearly outperformed by others. In particular, the combination of Il2rg + Itgb2 was identified among the best scoring candidate RG for every group of mice and every algorithm used in this experimental model. Finally, we have demonstrated that using "traditional" vs rationally-selected RG for normalization of gene expression data may lead to loss of statistical significance of gene expression changes when using large-scale platforms, and therefore misinterpretation of results. Taken together, our results highlight the need for a comprehensive, high-throughput search for the most stable reference genes in each particular experimental model.
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de la Laguna, La Laguna, Islas Canarias, Spain.
Yasmina E
YE
Hernandez-Santana
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de la Laguna, La Laguna, Islas Canarias, Spain.
Eduardo
E
Ontoria
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de la Laguna, La Laguna, Islas Canarias, Spain.
Ana C
AC
Gonzalez-García
Departamento de Biología, Facultad de Ciencias, Universidad Nacional de San Antonio Abad del Cusco, Cusco, Peru.
M Antonieta
MA
Quispe-Ricalde
Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Vicente
V
Larraga
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de la Laguna, La Laguna, Islas Canarias, Spain.
Basilio
B
Valladares
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de la Laguna, La Laguna, Islas Canarias, Spain.
Emma
E
Carmelo
J Biotechnol. 1999 Oct 8;75(2-3):291-5
10617337
Nucleic Acids Res. 2002 Jan 1;30(1):207-10
11752295
Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
12184808
Biotechnol Lett. 2004 Mar;26(6):509-15
15127793
Cancer Res. 2004 Aug 1;64(15):5245-50
15289330
Plant Physiol. 2005 Sep;139(1):5-17
16166256
BMC Mol Biol. 2006 Oct 06;7:33
17026756
Immunol Cell Biol. 2007 Feb-Mar;85(2):138-47
17146466
Genome Biol. 2007;8(2):R19
17291332
Vet Res. 2007 Jul-Aug;38(4):547-64
17540157
Biotechniques. 2008 Apr;44(5):619-26
18474036
Biochem Biophys Res Commun. 2008 Sep 12;374(1):106-10
18602371
Plant Cell. 2008 Jul;20(7):1734-5
18664615
Biochim Biophys Acta. 2008 Dec;1779(12):830-7
18790095
Immunobiology. 2009;214(2):101-11
19167988
Clin Chem. 2009 Apr;55(4):611-22
19246619
Biotechnol Adv. 2009 Jul-Aug;27(4):323-33
19472509
Parasitol Int. 2009 Dec;58(4):319-24
19698801
Anal Biochem. 2010 Apr 15;399(2):211-7
20036209
PLoS Pathog. 2010 Mar 12;6(3):e1000813
20300600
BMC Mol Biol. 2010 Mar 23;11:22
20331858
BMC Plant Biol. 2010 Mar 31;10:56
20353606
Biochem Biophys Res Commun. 2010 Sep 3;399(4):531-6
20678479
Mol Biotechnol. 2011 Jul;48(3):255-62
21184202
PLoS Pathog. 2011 Jan 06;7(1):e1001246
21253574
World Health Organ Tech Rep Ser. 2010;(949):xii-xiii, 1-186, back cover
21485694
Mem Inst Oswaldo Cruz. 2011 Aug;106(5):573-83
21894379
BMC Res Notes. 2011 Oct 14;4:410
21996334
PLoS One. 2011;6(11):e27592
22102912
PLoS Pathog. 2012 Jan;8(1):e1002417
22275864
Plant Mol Biol. 2012 Jan 31;:null
22290409
J Antimicrob Chemother. 2012 Aug;67(8):1905-14
22589456
PLoS One. 2012;7(5):e37517
22629413
PLoS One. 2012;7(6):e38489
22701652
J Immunol Methods. 2012 Oct 31;384(1-2):196-9
22884776
Mediators Inflamm. 2012;2012:949157
22933833
Vet Immunol Immunopathol. 2013 Jun 15;153(3-4):202-8
23545087
PLoS Pathog. 2013 Oct;9(10):e1003699
24204259
BMC Immunol. 2013 Nov 22;14:52
24267152
PLoS Negl Trop Dis. 2014 Jan 09;8(1):e2627
24421912
PLoS One. 2014 Feb 11;9(2):e88653
24523926
Int J Mol Med. 2014 May;33(5):1209-18
24626784
PLoS Negl Trop Dis. 2014 Apr 24;8(4):e2781
24762408
Muscle Nerve. 2015 Feb;51(2):276-81
24910410
Mol Genet Genomics. 2014 Dec;289(6):1157-69
24952483
PLoS Negl Trop Dis. 2014 Jul 10;8(7):e2914
25010815
BMC Res Notes. 2014 Sep 25;7:675
25256308
Plant Cell. 2014 Oct;26(10):3829-37
25361954
Immunobiology. 2015 Apr;220(4):437-44
25466588
Cell Rep. 2015 Feb 3;10(4):633-44
25620700
PLoS One. 2015 Jun 08;10(6):e0128934
26052705
J Neurotrauma. 2015 Nov 15;32(22):1777-88
26102571
Transplant Proc. 2015 Jul-Aug;47(6):2017-25
26293091
PLoS One. 2015 Aug 27;10(8):e0136499
26313760
Biomol Detect Quantif. 2014 Sep 06;1(1):23-33
27920994
Genome Res. 1996 Oct;6(10):986-94
8908518