title: Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).
abstract: We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), β-actin (ACTB), β-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, β-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.
authors.affiliation: National Dairy Research Institute, Karnal 132001, India.
authors.forename: N
authors.initials: N
authors.lastname: Varshney
authors.forename: A K
authors.initials: AK
authors.lastname: Mohanty
authors.forename: S
authors.initials: S
authors.lastname: Kumar
authors.forename: J K
authors.initials: JK
authors.lastname: Kaushik
authors.forename: A K
authors.initials: AK
authors.lastname: Dang
authors.forename: M
authors.initials: M
authors.lastname: Mukesh
authors.forename: B P
authors.initials: BP
authors.lastname: Mishra
authors.forename: R
authors.initials: R
authors.lastname: Kataria
authors.forename: S P
authors.initials: SP
authors.lastname: Kimothi
authors.forename: T K
authors.initials: TK
authors.lastname: Mukhopadhyay
authors.forename: D
authors.initials: D
authors.lastname: Malakar
authors.forename: B S
authors.initials: BS
authors.lastname: Prakash
authors.forename: S
authors.initials: S
authors.lastname: Grover
authors.forename: V K
authors.initials: VK
authors.lastname: Batish
mesh_terms: D000818:Animals; D002417:Cattle / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000235:genetics; D005796:Genes / Q000235:genetics*; D007774:Lactation / Q000235:genetics*; D008892:Milk / Q000166:cytology*; D019655:Quantitative Trait, Heritable; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary
chemical_list: D018076:DNA, Complementary