Evaluation and selection of internal reference genes from two- and six-row U.S. malting barley varieties throughout micromalting for use in RT-qPCR

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a popular method for measuring transcript abundance. The most commonly used method of interpretation is relative quantification and thus necessitates the use of normalization controls (i.e. reference genes) to standardize transcript abundance. The most popular gene targets for RT-qPCR are housekeeping genes because they are thought to maintain a static transcript level among a variety of samples. However, more recent studies have shown, several housekeeping genes are not reliably stable. This is the first study to examine the potential of several reference genes for use in RT-qPCR normalization during barley malting. The process of malting barley mechanizes the imbibition and subsequent germination of barley seeds under controlled conditions. Malt quality is controlled by many pleiotropic genes that are determined by examining the result of physiological changes the barley seed undergoes during the malting process. We compared the stability of 13 reference genes across both two-and six-row malting barleys (Conrad and Legacy, respectfully) throughout the entirety of the malting process. Initially, primer target specificity, amplification efficiency and average Ct values were determined for each of the selected primer pairs. Three statistical programs (geNorm, NormFinder, and BestKeeper) were used to rank the stability of each reference gene. Rankings were similar between the two- and six-row with the exception of BestKeeper’s ranking of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A consensus ranking among programs was determined using RefFinder. Our results show that Actin (ACT) and Heat Shock Protein 70 (HSP70) were the most stable throughout micromalting, while GAPDH and Cyclophilin (CYP) were the least stable. Two reference genes are necessary for stable transcript normalization according to geNorm and the best two reference genes (ACT and HSP70) provided a sufficient level of stability.