Abi 7300 rt pcr with sequence detection system v1

Primary cultures of strains were used for secondary culture and grown till an absorbance of 0.8 to 1 at 600 nm was reached. The cells were harvested, and RNA was extracted using the hot phenol method (76 (link)). Normalized RNA was used to synthesize the complementary DNA by iScript cDNA synthesis kit (catalog no.: 1708891; Bio-Rad). quantitative RT–PCRs of respective genes were performed with ABI-7300 RT–PCR with Sequence Detection System v1.4 (Applied Biosystems) according to the instructions by manufacturer using TB Green Premix Ex Taq II (catalog no.: RR820B; Takara). Relative mRNA expression was calculated according to the 2^−ΔΔCT method (77 (link)). The relative expression fold changes were calculated using the ACT1 gene as control. Data are represented as mean ± SD of three biological replicates.

The exponentially growing wild-type yeast (1588-4C) cells were left untreated or treated with VA (6 mM) for 3 h at 30 °C in SC liquid media and then cells were harvested. Total RNA was isolated by heat/freeze Phenol method as described earlier55 (link). 1 μg of DNA-free RNA was reverse transcribed to cDNA as per method supplied by iScript cDNA Synthesis Kit (Bio-Rad, India). Then PCR reaction was performed with primers of HAC1-S and ACT1 (See Table S2 for F/R primers). The PCR amplicon products were electrophoresed, stained with ethidium bromide, visualized and photographed. A representative image from at least two independent experiments for each condition was shown. For validation of microarray data, each qPCR reactions for selected genes were performed at least in duplicate on ABI-7300 RT-PCR with Sequence Detection System v1.4 (Applied Biosystems, CA) according to the conditions and protocol provided with VeriQuest SYBR Green qPCR Master Mix (Affymetrix, CA). Relative expression values (fold change) were calculated according to the ΔΔC_T method56 (link) using actin (ACT1) as a reference. Gene-specific primers used in this study were listed in Supplementary Table S2.

Primary cultures of strains were secondary cultured at 0.2 absorbance and grown till the exponential phase. The untreated/treated cultures were harvested, and RNA extraction was done using the hot phenol method [59 (link)]. RNA samples were normalized to the same concentration of 250 ng/μL to synthesize the complementary DNA using iScript cDNA synthesis kit (catalogue no.: 1708891; Bio-Rad). RT–PCRs of respective genes were performed using ABI-7300 RT–PCR with Sequence Detection System v1.4 (Applied Biosystems). Relative mRNA expression was calculated using the 2-ΔΔCT method [60 (link)]. The relative expression fold changes were calculated using control as the ACT1 gene. Data are represented as mean ± SD of three biological replicates. Complete list of primers is summarized in Table S2.