Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR.
BACKGROUND
Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR.


RESULTS
In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH.


CONCLUSIONS
Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues.
Nygard
Ann-Britt
AB
University of Copenhagen, Faculty of Life Sciences, Department of Basic Animal and Veterinary Sciences, Division of Genetics and Bioinformatics, Groennegaardsvej 3, 1870 Frederiksberg C, Denmark. abn@life.ku.dk
Jørgensen
Claus B
CB
Cirera
Susanna
S
Fredholm
Merete
M
D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D015870:Gene Expression; D009930:Organic Chemicals / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012313:RNA / Q000378:metabolism; D012016:Reference Values; D034421:Sus scrofa / Q000033:anatomy & histology / Q000235:genetics* / Q000378:metabolism
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D012313:RNA
Int J Biol Markers. 1999 Oct-Dec;14(4):247-50
10669954
Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
12184808
BMC Dev Biol. 2005;5:27
16324220
Biochem Biophys Res Commun. 2004 Jan 23;313(4):856-62
14706621
Lab Invest. 2005 Jan;85(1):154-9
15543203
J Mol Med (Berl). 2005 Dec;83(12):1014-24
16211407
Mol Cell Biochem. 2002 Jul;236(1-2):139-46
12190113
J Dairy Sci. 2006 Oct;89(10):4066-9
16960083
Genome Biol. 2007;8(4):R45
17407547
BMC Mol Biol. 2006;7:33
17026756
BMC Biotechnol. 2006;6:41
17026777
BMC Dev Biol. 2007;7:58
17540017
Thorax. 2002 Sep;57(9):765-70
12200519
BMC Mol Biol. 2005;6:4
15720708
Methods Mol Biol. 2000;132:365-86
10547847
BMC Vet Res. 2005 Sep 28;1:3
16188044
Mol Cell Probes. 2001 Oct;15(5):307-11
11735303
Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
12824337
BMC Biotechnol. 2006;6:24
16643647
Anim Biotechnol. 1998;9(1):67-78
9676236