qRT-PCR was used to validate qualification of RNA-seq for four genes (ST1, ST2, ST3 and ST4) and detected the transcript levels of the nine target genes with different FPKM values (ST1, ST2, ST11, ST12, ST21, ST31, ST32, ST39 and ST44) under low salinity and drought stresses. Gene-specific primers used for qRT-PCR were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) (Table 4 ).
qRT-PCR was performed on a Takara Thermal Cycle Dice™ Real Time System (Takara, Japan). A 10 μL qRT-PCR reaction contained 5 μL 2 × SPARKscript II RT Plus Master Mix (SparkJade Science Co., Ltd., China), 1 μL template, 0.2 μL of each of the forward and reverse primers (10 μM), and 3.6 μL ddH2O. Conditions used for qRT-PCR were as follows: 95 °C for 2 min 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s; and one cycle of 95 °C for 15 s, 60 °C for 60 s and 72 °C for 15 s. Three biological repeats and two technical replicates were performed. The relative transcriptional levels of the genes were calculated by the 2-ΔΔCt method [63 (link)], and β-actin was used as the internal reference [64 (link)].
qRT-PCR was performed on a Takara Thermal Cycle Dice™ Real Time System (Takara, Japan). A 10 μL qRT-PCR reaction contained 5 μL 2 × SPARKscript II RT Plus Master Mix (SparkJade Science Co., Ltd., China), 1 μL template, 0.2 μL of each of the forward and reverse primers (10 μM), and 3.6 μL ddH2O. Conditions used for qRT-PCR were as follows: 95 °C for 2 min 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s; and one cycle of 95 °C for 15 s, 60 °C for 60 s and 72 °C for 15 s. Three biological repeats and two technical replicates were performed. The relative transcriptional levels of the genes were calculated by the 2-ΔΔCt method [63 (link)], and β-actin was used as the internal reference [64 (link)].
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