Identifications and Validations of Reference Genes for Gene Expression Data Normalization of Chenopodium album

Abstract

Quantitative real-time PCR (qRT-PCR) is considered as an important technique for gene expression and validation analysis for several high-throughput proteomics and genomics data. However, reliable qRT-PCR results are dependent on appropriate reference genes used for data normalization under specific experimental condition and such information is limited in case of plants whose genome is not sequenced yet. Therefore, we sequenced and evaluated stability of seven novel internal control genes (Act, Act α, β-tub, 18SrRNA, GAPDH, EF1α and Ubq) of Chenopodium album, an important edible plant with medicinal values and have been recently used in synthesis as well as analysis of several gold and silver nanoparticles. Statistical algorithms like geNorm and NormFinder were used and compared for the samples treated with different types of stresses including, high/low temperatures, drought, heavy metal and salt stress. β-tubulin was revealed as the most stable genes under heat, drought and metal stress. While, 18SrRNA has shown the stable expression only under cold and salt stresses. Our data support the use of the combinations of two stable internal control genes for normalization of the gene expression. We compared the relative expression of a stress induced Chloroplast small Heat Shock Protein (Cp-sHSP) of C. album to further support our results. Together with all novel internal controls genes, this study also provides a platform for future gene expression studies of C.album using validated housekeeping genes.

ANALYSIS OF DNA SEQUENCE ENCODING GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) ON COCOR BEBEK (KALANCHOE x LAETIVIRENS)

“…A technique that can be used in the study of gene expression analysis is quantitative real time polymerase chain reaction (qRT-PCR) (Kozera & Rapacz, 2013). This technique requires the presence of reference genes as internal controls such as genes encoding actin, tubulin, ubiquitin, elongation factor-1α, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Libault et al, 2008;Basa et al, 2009;Chervoneva et al, 2010;Bao, 2016;Aman et al, 2017;Kosova et al, 2017;Singh et al, 2018). Generally, the internal control comes from the housekeeping gene cluster.…”