The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis ☆ Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus Justyna Jarczak a , Jarosław Kaba b , Emilia Bagnicka a, ⁎ a Department of Animal Sciences, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzębiec. Postępu 36A, 05-552 Magdalenka, Poland b Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Laboratory of Veterinary Epidemiology and Economics, ul. Nowoursynowska 166, 02-787 Warszawa, Poland abstract article info Article history: Received 14 October 2013 Received in revised form 23 July 2014 Accepted 24 July 2014 Available online 25 July 2014 Keywords: Goats Milk somatic cells Blood CAEV Housekeeping genes Normalization Real-Time PCR The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expres- sion in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The ex- pression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde- 3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large sub- unit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were consid- ered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of dis- ease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/ RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The properties of the PCR method are commonly used in the quanti- tative analysis of the transcript level. However, the level of target gene expression in changing conditions shows genetic instability and forces the choice of the best reference gene in a specific experimental model to validate the experiment by using related expression of target gene (Reboucas et al., 2013). Candidate reference genes should be selected Gene 549 (2014) 280–285 Abbreviations: ACTB, β-actin; B2M, β2 microglobulin; CLN3, battenin; DECR I, mitochondrial 2,4-dienoyl CoA reductase 1; EIF3K, eukaryotic translation initiation factor 3K; FPGS, folylpolyglutamate synthase; GAPDH, glyceraldehyde-3P-dehydrogenase; HPRT1, hypoxanthine phosphoribosyl-transferase I; MRPL39, mitochondrial 39S ribosomal protein L39; PGK, phosphoglycerate kinase; PPIA, cyclophilin A; PPIB, cyclophilin B; RNA18S1, 18S rRNA subunit; RPL13A, ribosomal protein L13A; RPL4, ribosomal protein L4; RPLP0, ribosomal protein large subunit P0; RPS15A, 40S ribosomal protein S15a; RPS9, 40S ribosomal protein S9; SDHA, succinate dehydrogenase complex subunit A; TBP, TATA box-binding protein; TOP2B, topo- isomerase II beta; TRAP1, triiodothyronine receptor auxiliary protein; TUBB, β-tubulin; UBQLN1, ubiquilin; UXT, ubiquitously expressed transcript protein; YWHAZ, zeta polypeptide; CAEV, caprine arthritis encephalitis virus; HKG, housekeeping genes; MSC, milk somatic cells; PFI, Polish Fawn Improved; PBMC, peripheral blood mononuclear cells; PWI, Polish White Improved. ☆ This research was conducted as part of the project “BIOFOOD — innovative, functional products of animal origin” No. POIG.01.01.02-014-090/09 co-financed by the European Union from the European Regional Development Fund under the Innovative Economy Operational Programme 2007–2013 and IGAB project S.IV.2. ⁎ Corresponding author at: Institute of Genetics and Animal Breeding, Polish Academy of Sciences in Jastrzębiec, ul. Postępu 36A, 05-552 Magdalenka, Poland. E-mail addresses: j.jarczak@ighz.pl (J. Jarczak), jaroslaw_kaba@sggw.pl (J. Kaba), e.bagnicka@ighz.pl (E. Bagnicka). http://dx.doi.org/10.1016/j.gene.2014.07.063 0378-1119/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene