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RNA Assumes More Prominent Role

Advances Unravel Complexities, Shed Light on Therapeutic Targets, and Move Molecule to Fore

• Kathy Liszewski

(Page 3 of 3)

Evolving Strategies

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.”

Tips for mRNA Capping

Capped RNAs can be made in vitro by incorporating cap analogs during transcription by enzymatic addition of cap structure after synthesis. Each method has advantages and drawbacks, according to G. Brett Robb, Ph.D., staff scientist at New England Biolabs:

• 5’-triphosphate transcripts can be capped using Vaccinia virus (VACV) mRNA capping enzyme with GTP and S-adenosyl methionine. This scalable approach allows for large amounts of RNA with m7GpppG cap structure to be generated.
• 5’-cap-specific labeling is possible using VACV. For high specific activity labeling, use the highest chemical concentration of radio-labeled GTP possible in the reaction.
• Co-transcriptional capping enables the incorporation of a variety of cap analogs including 3’-O-Me-m7GpppG (anti-reverse cap analog - ARCA), m7GpppG, GpppG, and ApppG.
• ARCA increases the yield of correctly capped mRNA since it incorporates in only one direction.
• Yields of co-transcriptionally capped mRNAs are influenced by many factors including concentration of NTPs and the ratio of cap analog to GTP. A ratio of 4:1 cap analog to GTP is suggested as a starting point.