Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

PLoS One. 2020 Aug 25;15(8):e0238189. doi: 10.1371/journal.pone.0238189. eCollection 2020.

Abstract

The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Animals
  • Bird Diseases / genetics*
  • Bird Diseases / metabolism
  • Cartilage, Articular / metabolism*
  • Chickens / genetics*
  • Chickens / metabolism
  • Epiphyses, Slipped / genetics
  • Epiphyses, Slipped / metabolism
  • Epiphyses, Slipped / veterinary*
  • Femur
  • Gene Expression
  • Gene Expression Profiling / statistics & numerical data
  • Gene Expression Profiling / veterinary
  • Male
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction

Substances

  • RNA, Messenger

Grants and funding

IRS was supported by a Graduate Teaching Assistant Scholarship (PROMOP)/Udesc #18001/2020, Brazil. MRS received a scholarship from the Coordination for the Improvement of Higher Education Personnel/The Santa Catarina State Research Foundation (CAPES/FAPESC) #0337/2018, Brazil. DEPM is a recipient of a PIBIC scholarship #135359/2018-0 from the National Council of Scientific and Technological Development (CNPq) at Embrapa Swine and Poultry National Research Center, Brazil. MCL is recipient of a productivity fellowship #307026/2017-5 from CNPq. This study was supported by project # 01.11.07.002.04.03 from the Brazilian Agricultural Research Corporation (EMBRAPA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.