Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries
Authors: Song Yashu , Hanner Robert H. , Meng Baozhong 
Published: October 2021

Abstract
Background
Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water.

Results
We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change.

Conclusions
We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.