Revertaid m mulv reverse transcriptase kit

First-strand cDNA synthesis was carried out using RevertAid M-MuLV Reverse Transcriptase kit (Fermentas) as per the instructions of the manufacturer. Nearly 1 μg purified RNA acting as a template, was incubated with random hexamers and denatured at 65 °C for 5 min followed by snap chilling. Other reaction components were subsequently added to the tube in 20 μl final volume. The tube was incubated in a thermal cycler at 25 °C for 5 min, 45 °C for 1 h and 70 °C for 5 min, consecutively. Along with the cDNA reaction, a negative control reaction, termed RT-, was also included from which RT enzyme was omitted to rule out the presence of any contaminating genomic DNA. PCR reactions were performed with 1 μl template from each reaction (cDNA rxn, RT- rxn). In parallel with these reactions, another positive control PCR reaction was set up from gDNA template. The PCR products were loaded on an agarose gel followed by visualization on a UV transilluminator.

Total RNA was extracted from livers as well as left tibias and femurs of 24-week-old mice using TRIzol reagent (Invitrogen), according to the manufacturer’s protocols. After digestion with DNase I (Qiagen, Antwerp, Belgium), cDNA was synthesized from 1 μg RNA using the RevertAid M-MuLV Reverse Transcriptase kit (Fermentas, St Leon-Rot, Germany) and random hexamer primers (Fermentas). The PCR reaction mixtures (10 μl) contained 1× Fast SYBR Green qPCR Master mix (ThermoFisher Scientific) and 0.15 μL (2.5 μM) of each primer. The StepOne Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) was used. The primers used were: human SHBG-Fw 5′-ATCACAAAAACCTCCTCCTCCTT-3′. SHBG-Rev 5′-ATCTCCCATCATCCAGCCGT-3′ (190 Bp amplicon), mouse Cyp19a1-Fw 5′-TGGCAAGCTCTCCTCATCAA-3′, Cyp19a1-Rev 5′-TCTCCACGTCTCTCAGCGA-3′ (200 Bp amplicon), mouse 18S-Fw 5′-CGCCGCTAGAGGTGAAATTC-3′, 18S-Rev 5′-TTGGCAAATGCTTTCGCTC-3′. All primers were designed using NCBI Primer-BLAST software to hybridize to different exons and generate a single amplicon in melting curve assays. Gene expression was quantified using the relative (ΔΔCT) method with normalization to the levels of 18S ribosomal RNA.

To detect mRNA levels of tissue factor, TFPI-1 and TFPI-2 in ovaries and preovulatory follicles after hCG treatment (0, 3, 6, 9 and 12 h), total RNA was extracted using TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH, USA), according to the manufacturer’s instructions. Ten or twenty micrograms of total RNA was reverse-transcribed using the RevertAid M-MuLV reverse transcriptase kit (Fermentas, St. Leon-Rot, Germany) to evaluate gene expression. Real-time PCR was then performed on a Rotor-Gene Q 5plex (QIAGEN, Hilden, Germany), located at Korea Basic Science Institute (Gwangju, Korea), using the QuantiTect SYBR Green PCR Kit (QIAGEN) at 95°С for 20 s, 60°С for 20 s, and 72°С for 30 s. Specific primers were designed using the PRIMER3 software (Table 1). The average Ct value in triplicate for each gene was divided by the linear Ct value of β-actin to obtain relative abundance of the transcripts. β-Actin was used as an internal control for all measurements.
Table 1

PCR primers used to obtain cDNAs for rat genes

F Forward, B Backward

Expression analysis using real-time reverse transcriptase PCR (RT-PCR) was performed as previously described [10 (link)]. Exponential-phase S. maltophilia strains were challenged with the tested substances at sub-minimum inhibition concentrations i.e.100 μM H₂O₂, 50 μM cumene hydroperoxide, 50 μM NEM, 200 μM menadione, 100 μM plumbagin, 100 μM paraquat, 500 μM diamide, 32 μg/ml acriflavine, 200 μM phenazine, and 8 μg/ml benzalkonium chloride for 30 min before the cells were harvested for total RNA extraction. Reverse transcription reaction was performed using 5 μg of DNase I-treated RNA, random hexamers, and the RevertAid M-MuLV reverse transcriptase kit (Fermentas, Latvia). RT-PCR was performed using 20 ng of cDNA, an mfsC specific primer pair (BT4203 and BT4204 [Table 1]), and SYBR green PCR master mix (Applied Biosystems, Thermo Fisher Scientific, USA). The 16S ribosomal RNA gene amplified using primers BT2781 and BT2782 (Table 1) was used as the normalizing gene. The PCRs were run on an Applied Biosystems StepOne Plus for 40 cycles under the following conditions: denaturation at 95°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s. The relative expression was calculated using StepOne software v2.1 and expressed as fold expression over the uninduced level of K279a. For each experiment, at least three independent biological repeats were performed.