Realmastermix sybr green

The expression levels of selected genes were measured by real-time quantitative RT-PCR analysis. Three or more reactions were performed for each sample using a RealMasterMix (SYBR Green) kit (TianGen, Beijing, China) with gene-specific primers (Additional file
23: Table S3) on a Rotor-Gene Q (QIAGEN, Valencia, CA). The RNA quantity was normalized to the ACTB content, and gene expression was quantified according to the 2^-ΔCt method.

For the RNA-seq analysis, the wild-type XL280 strain and isogenic Pum1 overexpression mutants were cultured in YPD liquid medium (extremely mating-suppressing condition) at 30°C overnight. The overnight culture was then washed with cold water and dropped on V8 agar (pH = 7) for mating induction. The cells were collected at different time points post mating stimulation for the isolation of total RNA. The total RNA was extracted using TRIzol Reagent (CW0580M, CWBIO) and an Ultrapure RNA Kit (CW0581M, CWBIO) according to the manufacturer’s instructions. Total RNA (2 μg) was subjected to gDNase treatment, and single-stranded cDNA was synthesized by a Fastquant RT Kit (with gDNase, KR106-02, Tiangen) according to the manufacturer's instructions. The relative mRNA level of selected genes was measured by real time RT-PCR using RealMaster Mix (SYBR Green, FP202-02, TIANGEN) in a CFX96 TouchTM Real-time PCR detection system (Bio-Rad). The primers used for qPCR in this study are listed in the Key Resources Table. The relative transcript levels were normalized to those of the reference housekeeping gene TEF1 and determined using the 2^-ΔΔCT approach.

Total cellular RNA was extracted from E. coli strain harboring σ⁷⁰ mutant C9 grown overnight with or without 38.0 % (v/v) cyclohexane using the Simply P Total RNA Extraction Kit (BioFlux, Japan). Reverse transcription step was carried out using RevertAid First Strand cDNA Synthesis Kit (Thermo, USA) with random primer mix following the manufacturer’s manual. Real-time quantitative reverse transcription PCR (RT-qPCR) was performed with RealMasterMix (SYBR Green) (TIANGEN, China) using Bio-Rad iQ5 real-time PCR detection system (Bio-Rad, USA). The bacterial 16S rRNA gene sequence was used as a reference gene in real-time PCR (Additional file 6: Table S3). The real-time PCR conditions were as follows: 1 min at 94 °C, 35 cycles at 94 °C for 10 s, followed by 55 °C for 30 s and 68 °C for 15 s. To analyze the gene expression level, ΔΔCt method was chosen and the standard curves of each primer were plotted to ensure similar amplification efficiency compared with the reference gene.

The 5 x 10⁵ cells were seeded in each well of 24-well culture plate. Cells were collected with TRIzol (Takara) 48 h post-transfection. The neuroretina of RCS rat, MNU-treated rat, and control rats were also collected with TRIzol. For detail, under a dissection microscope, the anterior segments of the eye were removed. Then the eye cup was radially cut into 3 to 4 pieces from periphery to the optic nerve head. Each piece was carefully dissected and neuroretina was isolated [42 (link)].
Total RNA was extracted and reverse transcription was performed using Reverse Transcriptase M-MLV RNase H^- (Takara). Real-time PCR was performed in a Bio-Rad CFX Connect Real-time System by using RealMasterMix (SYBR Green) (Tiangen Biotech, China). PCR amplification was performed in duplicate (program: denaturation at 95°C for 15 min, followed by 40 cycles of 95°C for 10 s, 60°C for 40 s). All primer sequences are listed in S2 Table.

The total RNA was homogenized in the TRIzol reagent; then, the total RNAs were extracted following the manufacturer’s protocol. After determining the concentration of RNAs with an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), quantitative real-time PCR was performed in the PCR system (Pikoreal 96, Thermo Fisher) with Real Master Mix SYBR Green (FP202, Tiangen, Beijing, China) following the manufacturer’s instructions. The gene-specific primers are listed in Table 1. The relative expression of each gene was expressed as fold-changes with respect to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Total RNA of the prefrontal cortex and unilateral hippocampus were isolated using Trizol (Invitrogen), and cDNA was reverse-transcribed from total RNA (2 μ g) using PrimeScript® RT reagent Kit With gDNA Eraser (Perfect Real Time) (Takara), following the manufacturer's instructions. Real-time PCR was performed using the RealMasterMix (SYBR Green) (Tiangen). Specific primers were designed for genes, while the housekeeping gene β-actin was chosen as a control for normalizing the relative mRNA level (the primer sequences are available upon request). Twenty μL reaction agents were carried out in the Mx3005P quantitative PCR system (Stratagene, La Jolla, CA, USA) comprised of 9 μL of 2.5 × RealMasterMix/20 × SYBR solution, 1 μL of template cDNA, 0.5 μM of each primer, and 9 μL sterile water. Negative controls containing no template were also performed for each primer pair. The thermal cycling conditions were as follows: 95°C for 2 min followed by 40 cycles of 95°C for 20 s, 60°C for 20 s, and 68°C for 40 s. The melting curve analysis was performed to eliminate the presence of unspecific products by a high-resolution data collection during an incremental temperature change from 55 to 95°C with a ramp rate of 0.2°C/s. The data derived from the Mx3005P quantitative software were calculated using the 2^−△C_T formula (Livak and Schmittgen, 2001 (link); Wang et al., 2006 (link)).

cDNA was synthesized using Reverse Transcriptase XL (AMV) (TaKaRa, Japan) according to the manufacturer’s protocol in a 20 μL reaction system. The reverse transcription reaction mixture contained 5 μL total RNA (1 μg), 1 μL of each 10 mM dNTPs, 1 μL of random primer (9 mer) (50 μM), 1 μL oligo d(T)₁₈ primer (50 μM) (TaKaRa, Japan), and 6 μL DEPC water. The mixture were incubated at 65°C for 5 min and cooled on ice for 5 min, then 4 μL 5× Reverse Transcriptase buffer, 1 μL RNasin (TaKaRa, Japan), and 1 μL AMV (5 U) were added. The mixture was incubated at 37°C for 2.5 h, and then the enzyme was inactivated by incubating at 72°C for 15 min. qPCR was carried out in an iQ5 Real Time PCR Detection System (BioRad, USA) with RealMasterMix SYBR Green (TIANGEN, China). Primers used for validation of differentially expressed genes are shown in S1 Table. All data were normalized with the level of the Fv26S internal transcript control. Relative fold changes in genes expression were calculated using the comparative Ct (2^-ΔCt) method. Each sample was quantified in triplicate.