Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits

J Agric Food Chem. 2012 Jul 11;60(27):6855-65. doi: 10.1021/jf300419w. Epub 2012 Jun 29.

Abstract

Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chemical Fractionation / methods*
  • Fruit / chemistry
  • Fruit / genetics*
  • Fruit / metabolism
  • Gene Expression Regulation, Plant
  • Olea / chemistry
  • Olea / genetics*
  • Olea / metabolism
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Plant / genetics
  • RNA, Plant / isolation & purification*
  • RNA, Plant / metabolism
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards

Substances

  • Plant Proteins
  • RNA, Messenger
  • RNA, Plant