Fast universal sybr green master

Total RNA was extracted from vein tissues and cell samples and the complementary DNA was synthesized using a RevertAid first strand cDNA synthesis kit (Thermo Scientific, MA, USA). Detected via qRT-PCR; gene expression levels were performed on a Light Cycler^® 480 System (Roche, Basel, Switzerland) using Fast Universal SYBR Green Master (Roche, Basel, Switzerland). Primer Analysis Software (Oligo 7.24, Molecular BiologyInsights, Inc., USA) was used to design specific oligonucleotide primers. These mRNA and LncRNA primers were commercially synthesized by Beijing Genomics Institute Co., Ltd., China. GADPH was the housekeeping gene used as an internal reference. The mRNA and LncRNA relative abundance for each gene was calculated according to the method of 2^−ΔΔCt, accounting for gene specific efficiency and was normalized to the mean expression of GADPH.

WT yeast cells were grown to 10⁵ CFU/mL in YPD medium and then treated with 25 μg/mL compounds or 0.25% DMSO for 16 hours at 30 °C. Then, the cells were collected by centrifugation at 1500 g for 5 min and washed twice with water. Total RNA was prepared using the Yeast RNA kit (OMEGA R6870-01, Norcross, GA, USA) and treated with DNase Ⅰ (TransGen GD201-01, Beijing, China) to remove genomic DNA. The resulting RNA was reverse-transcribed with the cDNA synthesis super-mix kit (TransGen AT311, Beijing, China). qRT-PCR was performed using Fast Universal SYBR Green Master (Rox, Roche, Mannheim, Gemany) and gene-specific primers (Table S1). The DNA level was measured using an Real-Time Quantitative Thermal Cycler (FTC-3000, Funglyn Biotech Inc, Toronto, Canada). The expression levels were correlated with the copy numbers used to amplify the gene to reach the threshold, then represented as the mean ± SEM relative to the cells treated with DMSO. One-way ANOVAs were performed to identify deviation from the mean.

Total RNA was isolated from tissue samples using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). The RNA concentrations were determined using the GeneQuant 1300. The procedure for reverse transcription was performed according to the manufacturer's instructions (Roche, Basel, Switzerland). Synthesized cDNA was stored at −20°C for PCR. Oligo 6.0 software was used to design specific primers based on known sequences (Table 1). Chicken β-actin was used as a housekeeping gene and an internal reference. Primers were synthesized by Invitrogen Biotechnology Co. Ltd. in Shanghai, China.
QRT-PCR was performed on a LightCycler^®480 Detection System (Roche, Basel, Switzerland) using Fast Universal SYBR Green Master (Roche, Basel, Switzerland). The program was run as follows: 1 cycle at 95°C for 30 s followed by 40 cycles at 95°C for 5 s and at 60°C for 34 s. Dissociation curves for each PCR reaction were analyzed by using the Dissociation Curve 1.0 software (Applied Biosystems) to detect and eliminate possible primer-dimers and non-specific amplifications. The relative abundance of mRNA was calculated according to the method of Pfaffl [60 (link)].