Abstract
Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4 A, Elongation factor-1 alpha, FTSH protease 4, U2 auxiliary fator, Succinol Co-enzyme A, Tubulin alfa-5, Tubulin beta-6, Ubiquitin conjugating enzyme. Total RNA was extract from leaf tissues of U. brizantha subjected to 6, 12 and 24 h of cold and heat stresses (10 and 45 °C, respectively), and drought, including moderate (−0.5 to −0.7 MPa), severe (−1.1 to −1.8 MPa) and recovery after re-watering. The RefFinder web-based tool was used to rank the most stable reference genes for each stress. Elongation factor-1 alpha, Elongation factor-1 alpha or Ubiquitin conjugating enzyme, and Eukaryotic initiation factor 4 A were the most stable genes for heat, cold and drought stress, respectively. The expression of Rubisco large subunit gene was normalized against the most stable gene selected by ReFfinder for each stress.
Highlights
Quantitative real-time PCR (RT-qPCR) is considered the most accurate and reliable technique to measure gene expression and to validate data obtained by other methods like cDNA microarrays and RNA-seq
We evaluate the suitability of nine reference genes [Actin 12 (ACT12), Eukaryotic initiation factor 4 A, Elongation factor 1-alpha (EF1-α), FTSH protease 4 (FTHS), U2 auxiliary fator (U2AF), Succinol Co-enzyme A (SucCoa), Tubulin alfa-5 (α-TUB5), Tubulin beta-6 (β-TUB6) and Ubiquitin conjugating enzyme (UbiCo)] as candidates for normalization in RT-qPCR assays to study the transcriptional changes involved in the responses of U. brizantha to three different abiotic stresses using the RefFinder web-based tool[8]
The melting curve analysis performed at the end of RT-qPCR reactions for all selected candidate genes to check the specificity and integrity of the PCR products showed the presence of a single peak (Fig. 1)
Summary
Quantitative real-time PCR (RT-qPCR) is considered the most accurate and reliable technique to measure gene expression and to validate data obtained by other methods like cDNA microarrays and RNA-seq. NormFinder[4], geNorm[5], BestKeeper[6] and ΔCt method[7] are the most commonly methods used for this propose These softwares calculate a measure of the stability of potential reference genes by comparing their individual stability in relation to the other tested genes under different experimental conditions. We evaluate the suitability of nine reference genes [Actin 12 (ACT12), Eukaryotic initiation factor 4 A (eIF4a), Elongation factor 1-alpha (EF1-α), FTSH protease 4 (FTHS), U2 auxiliary fator (U2AF), Succinol Co-enzyme A (SucCoa), Tubulin alfa-5 (α-TUB5), Tubulin beta-6 (β-TUB6) and Ubiquitin conjugating enzyme (UbiCo)] as candidates for normalization in RT-qPCR assays to study the transcriptional changes involved in the responses of U. brizantha to three different abiotic stresses using the RefFinder web-based tool[8]
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