Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk

Abstract

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells.

Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells

“…in the present study) the optimal pair does not include the most stable gene. It may be related to the necessity to compensate for the overexpression of the one reference gene by another one, which is underexpressed in the same group [ 26 ]. Detailed results regarding the intra- and intergroup variation of investigated reference genes calculated by NormFinder are shown as supplementary data ( S4 Fig ).…”

Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts

“…The changes in the subpopulation fraction potentially affect the choice of the most suitable reference gene. Accordingly, several studies reported the necessity of finding a suitable reference gene for studying gene expression in milk somatic cells in various species such as goats ( Modesto et al, 2013 ; Jarczak et al, 2014 ), zebu ( Varshney et al, 2012 ), or yaks ( Bai et al, 2014 ). The variation in the subpopulation fraction can also be a problem when using a technique such as RNA Sequencing as it takes into account all the genes expressed in a given sample.…”

Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

“…In addition, the amount of RNA maybe insufficient at times and accurate computation of cell number is often infeasible. Another proposed strategy is to use reference genes in data normalization; this strategy is currently considered a robust approach in most cases [ 8 ]. Reference genes are supposed to be constitutively expressed, and their expression should remain stable irrespective of experimental conditions such as developmental stage, experimental treatment, physiological state and tissue type [ 9 , 10 ].…”