Myiq2 real time pcr detection system

Bacterial pellets of 5 ml of microaerobic cultures in LB medium supplemented with KNO₃ were used for total RNA extraction using Total RNA Extraction Kit (RBC Biosciences). After treatment with DNaseI, cDNA was obtained using random hexamers (Promega) and Revert Aid Reverse Transcriptase (ThermoFisher Scientific, Waltham, USA) following the manufacturer’s instructions. qRT PCR was performed using a MyiQ2 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, USA) and Real Time PCR mix (EvaGreen qPCR Mix Plus, no ROX, Solis Biodyne). The cycling conditions were as follows: denaturation at 95 °C for 5 min, 40 cycles at 95 °C for 25 s, 58 °C for 15 s, and 72 °C for 15 s, with fluorescence acquisition at 80 °C in single mode. For normalization 16S rRNA gene was used and relative changes in the expression of anr gene for microaerobic conditions was obtained through the relative standard curve method. Oligonucleotides used are detailed in Table S4.

Total RNAs were extracted from the testis of B. dorsalis using TRIzol reagent (Invitrogen, CA, USA) and cDNA was synthesized using 1 μg total RNA and miScript Hiflex buffer, in a final volume of 20 μL of miScript II reverse transcriptase reaction (Qiagen, Valencia, CA) according the manufacturer’s protocol. Modified oligo-dT primers with 3′ degenerate anchors and 5′ universal tag sequences were used for the specific synthesis of cDNA of mature miRNA and mRNA. miRNA primers (Table 1) for qPCR were designed using the miScript miRNA product design webpage (Qiagen, Valencia, CA). Primers for bmfrn, actin and U6 cDNA (Table 1) were designed using Primer Premier 5.0 software (Premier, Canada). qRT-PCR was made up of 2.5 μL of 10x diluted cDNA, 12.5 μL of SYBR Green Master Mix (miScript SYBR Green PCR Kit, Qiagen Valencia, CA), 2.5 μL of 10 mM of forward and reverse primer and 5 μL of RNase free water in a 25 μL total volume. qRT-PCR was conducted on MyiQ2 real time PCR Detection System (BioRad) with the following thermal profile: 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. Three technical replicates per sample were conducted and relative expression levels were calculated using 2^−△△Ct method35 . Expression of actin and U6 were used to normalized the expression of mRNA and miRNA respectively14 (link)36 (link)

Total RNA was extracted from control and treatment group HK-2 cells using Trizol reagent and quantified using Nanodrop Spectrophotometer (Thermo Scientific). Gene expression changes were analyzed by single step qRT-PCR amplifications in MyiQ2 real time PCR detection system (BioRad Laboratories, Hercules, CA) using one-step RT-PCR kit with SYBR green and total RNA (200ng). PCR reactions were performed with reverse transcription at 50°C for 15 min, denaturation and reverse transcriptase inactivation at 95°C for 5 min, followed by 40 cycles (10 seconds each) of denaturation at 95°C and annealing and extension at 60°C for 30 seconds. Melt curve analysis was included to confirm the specificity of PCR products. PCR amplification data of each gene were normalized to Ct value of internal housekeeping gene (GAPDH) from the same sample and the fold-changes in gene expression were calculated by using the delta-delta Ct method [87 ]. List of forward and reverse primer sequences used for RT-PCR analysis have been given in Table 1.

The tissue samples stored in RNAlater (Invitrogen, Carlsbad, CA) were homogenized by using the Fast Prep 24G instrument (MP Biosciences, Santa Ana, CA). Total RNA was prepared using the TRIzol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA) and single-strand cDNA was synthesized from the RNA in a reaction mixture containing optimum blend of oligo(dT) primers and iScript reverse transcriptase (Bio-Rad, Richmond, CA). qRT-PCR amplifications were performed using rEVAlution 2x qPCR Master Mix (Empirical Bioscience, Grand Rapids, MI) in an MyIQ2 Real-Time PCR Detection System (Bio-Rad, Richmond, CA) following manufacturer's protocol. To determine the specificity of amplification, melting curve analysis was applied to all final PCR products. The relative amount of target mRNA was calculated by the comparative threshold cycle method by normalizing target mRNA threshold cycle to those for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used for analysis were as follows: NQO1: sense primer, 5’-AGGATGGGAGGTACTCGAATC-3’, anti-sense primer, 5’-AGGCGTCCTTCCTTATATGCTA-3’; GAPDH: sense primer, 5’-CTTCACCACCATGGAGAAGGC-3’, anti-sense primer, 5’-GGCATGGACTGTGGTCATGAG-3’.