Examining gene expression via RNA analysis is a field on the move. Alice Lan, Ph.D., post-doctoral fellow in the laboratory of Donald R. Love, Ph.D., School of Biological Sciences, University of Auckland, provided a perspective on studies she performed previously on zebrafish.
“Over the years, whole mount in situ hybridization (WISH), another method of examining RNA transcripts, has been widely used among zebrafish researchers. Although WISH gives spatial patterns (in what tissues the transcripts are expressed), it is generally less sensitive and only semiquantitative (when coupled with imaging-analysis software). It is a usually a three-day protocol.
“However, qRT-PCR is a rapid, sensitive, and quantitative method for transcript analysis. It also allows multiplexing of differentially labeled probes so multiple transcripts can be examined at the same time.
“To examine expression during different stages of development simply requires extracting RNA from larvae at different time points. This is followed by cDNA synthesis and qPCR. The challenge is that one needs to validate the qRT-PCR results with another method. I think qRT-PCR and WISH experiments complement each other nicely.”
Dr. Lan is now applying her knowledge of RNA analysis to human clinical genetics. “We often need to correlate genetic variants (DNA) with gene expression.
“First, we have been emphasizing the use of multiple reference genes, rather than one reference gene, because how do you determine that a particular reference gene is stable without comparing it to other genes? Secondly, whenever possible, especially qPCR with SYBR Green, the amplicon should be sequenced to confirm their identity.
“Thirdly, disclosing detailed descriptions of RNA extraction protocol and data analysis is particularly important as the quality of RNA can greatly influence the efficiency of cDNA synthesis and qPCR. In terms of data analysis, readers should be able to easily figure out how the final ‘expression units’ are derived rather than second guessing what has been done to raw data.”