Normalizing TaPDIL1-1 Expression via Reference Genes

To determine how the adoption of different reference genes can affect the normalization of the expression data for a gene of interest, the same 24 plant cDNA samples used for the stability analyses of reference genes (see Plant material) were also analysed by qRT-PCR for the expression of TaPDIL1-1 (a gene encoding the Protein Disulfide Isomerase). Isolation and characterization in wheat of the three homoeologous gene sequences encoding PDI (TaPDIL1-1) and of their full-length transcripts have been reported previously [40 (link)]. The primer pair used for TaPDIL1-1 qRT-PCR analysis (forward: 5'-CGTGGTCTTCAAATCCTG-3'; reverse 5'-GTAACCCTGGACATCAAAC-3') was designed in conserved regions of the three homoeologous cDNA sequences at their 3' ends (Accession numbers: AJ868105, AJ868106, AJ868107) with annealing temperature of 55°C. The PCR efficiency was 100.35 ± 0,365 with a coefficient of determination (R²) of 0,998; the amplicon size was 187 bp. The TaPDIL1-1 expressions were normalized using five different strategies: 1) geometric average of the three references genes selected as the most stable genes by geNorm; 2) geometric average of the two references genes selected as the most stable genes by NormFinder; 3) the single most stable gene identified by NormFinder; 4–5) the two genes related to the Unigene clusters Ta54825 (Actin) and Ta25534 (α-tubulin) used alone. Normalized TaPDIL1-1 relative values are given as mean value ± SD. Standard deviations on normalized expression levels were computed according to the geNorm user manual [47 ].