Validating RNA-Seq Expression Levels via qRT-PCR

Twelve transcripts were randomly selected for real-time quantitative PCR (qRT-PCR) to verify the accuracy of the levels of expression obtained under RNA-seq. Genes included: GMPM1: 18 kDa seed maturation; PP2C-51: protein phosphatase 2C 51-like; LEA-DC3: late embryogenesis abundant protein Dc3-like; DH1a: dehydrin DH1a; ATHB22: homeobox leucine zipper; SUS2: sucrose synthase 2-like; PIP2-2: aquaporin PIP2-2-like; XTH6: xyloglucan endotransglucosylase/hydrolase protein 6; GOLS2: galactinol synthase 2-like; CuSOD1: Superoxide dismutase [Cu-Zn]; APX_Chl: chloroplast ascorbate peroxidase. All primer sequences are presented in Table S1. The primers were designed using Primer3 web version 4.1.0 [84 (link)] with an e-value < 2 × 10⁻⁴ and a score >41. cDNA was synthesized from 1 μg total RNA using the SensiFASTTM cDNA Synthesis kit (Meridian BioScience, Cincinnati, OH, USA), according to the manufacturer’s recommendations. The presence of a single amplification product of the expected gene size was verified by electrophoresis on a 1.5% agarose gel. PCR reactions were prepared using the SensiFASTTM SYBR No-ROX kit (Meridian BioScience, USA) according to the manufacturer’s protocol. One negative sample was included for each primer pair, in which cDNA was replaced by water. Reactions were carried out in 96-well plates using a qTOWER 2.2 Thermal Cycler (Analytik, Jena, Germany) using the following parameters: hot start activation of the Taq DNA polymerase at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, elongation at 72 °C for 30 s. A melting curve analysis was performed at the end of the PCR run by a continuous fluorescence measurement from 55 °C to 95 °C with sequential steps of 0.5 °C for 15 s. A single peak was obtained and no signal was detected in the negative controls. Three technical replicates were used for each analyzed plant. Gene expression was quantified using malate dehydrogenase (MDH) and ubiquitin (UBQ10) as reference genes [85 (link)]. To understand the agreement of these results with the one from RNA-sequencing, heatmaps were constructed considering the levels of transcripts and their expression levels from qRT-PCRs after being log₂ FC scaled by gene expression across treatments.