Relative Quantification Of Gene Expression Using QRT-PCR Analysis

However relative quantification of gene expression using qRT-PCR is highly influenced by the expression stability of internal control or reference genes used for transcript normalization of target genes. The use of inappropriate or unstable reference gene(s) can seriously impact the transcript quantification results leading to false inferences or misinterpretations21,22. Accurate normalization is thus necessary for obtaining biologically meaningful expression data and hence qRT-PCR analysis greatly depends upon careful selection of reliable reference gene(s) which should be stably expressed across various tissue samples, developmental stages and experimental conditions23,24. Numerous studies have been carried out until now in various economically
Hence considering the inevitability of crop- as well as stress-specific internal control genes for accurate normalization of transcripts in qRT-PCR, the present study was carried out to evaluate the expression stability of 10 candidate reference genes in pearl millet subjected to individual or combination of abiotic stresses and or abscisic acid (ABA), a stress hormone at early- (1h) and late- (24h) stress durations as well as in different developmental tissues of two contrasting
The Ct values were monitored under four groups including all samples, individual abiotic stresses, multiple abiotic stresses, and developmental tissue samples. The Ct values of 10 potential reference genes ranged from 15.5 to 34.7 (Table 2). In the all samples set, the mean Ct values showed a minimum of 21.7 ± 3.4 and maximum of 28.9 ± 2.5 for highest and lowest expression levels for RPL20 and TIP41, respectively. RPL20 and TIP41 also showed minimum and maximum average Ct values, respectively in individual stress sample set as well as in multiple stress samples set. On the other hand in the developmental tissue set, UBC-E2 has minimum average Ct value of 21.1 ± 4.8 while TIP41 has maximum average Ct value of 29.4 ± 3.7. Further CV of the Ct values was also calculated to evaluate the expression levels of candidate reference genes under all four experimental sets, where lower values represent lower variability or maximum stability. The CV of 10 reference genes among all samples ranged between 4 to 15% (Fig.). EF-1 was the least variable reference gene with a CV of 4.5% among 10 candidate reference genes studied and MDH was the most variable with a CV of 15.7%. The stability ranking of all candidate reference genes on the basis of CV values is