One step sybr primerscript rt pcr kit 2

Vibrio cholerae strains were grown in LB medium to an OD₆₀₀ of 0.6. Total RNA was extracted from the culture of C6706 and other test strains using the SuperScript™ III Reverse Transcriptase and DNA-free™ DNA Removal Kit (Thermo Fisher). The RNA samples were analyzed by quantitative real-time reverse transcription-PCR (qRT-PCR) using the One Step SYBR Primerscript RT-PCR Kit II (TaKaRa). Relative expression values (R) were calculated using the equation $\text{R}=2^{-\left(\mathrm{\Delta }Cqtarget-\mathrm{\Delta }Cqreference\right)}$ , where Cq is the fractional threshold cycle. recA of C6706 was used as an internal reference. Run the qRT-PCR as follows: Pre-incubation (1 cycle): 95 °C for 1 min; Amplification (40 cycles): Denaturation at 95 °C for 10 s, Annealing at 60 °C for 30 s (to collect fluorescence signals); Melting (1 cycle): 95 °C for 10 s, 65 °C for 30 s, and 97 °C for 1 s; Cooling (1 cycle): 37 °C for 30 s. A control mixture using total RNA as a template was performed for each reaction to exclude chromosomal DNA contamination. The primers used for these target genes, recA and hlyA are listed in Additional file 1: Table S2.

Cultures inoculated from single colonies (2 ml, three replicates; strains IE01 and DB01) were grown overnight in LB medium at 37 °C and 170 rpm. Cultures were then diluted 1:200 in M9 minimal medium, and were grown for 2 h at the same conditions. Cells were pelleted by centrifuging 2 min at 13,000 rpm, and resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Cells were broken with phenol-chloroform and vortexing thoroughly, recovering the RNA in the aqueous phase. Samples were then passed through a silica-based, DNA-clean column (Zymo), and were eluted in TE buffer with application of DNase I (Fermentas). Total RNA eluted was quantified in a NanoDrop.
One-step SYBR PrimerScript RT-PCR Kit II (Takara) was used for detection, following the Kit protocol for preparing the reaction volumes. 16S ribosomal RNA (rRNA) was used as housekeeping gene to normalize RNA quantity in each reaction. The primer sequences for amplifying marA and 16S rRNA were taken from previous work.^{53 (link),54 (link)} Reactions in triplicate were carried out using a Step One Plus Real-Time PCR System (Applied Biosystems). The thermal cycling program for amplification was 5 min at 42 °C, 10 s at 95 °C, and 40 cycles of 5 s at 95 °C and 34 s at 60 °C (Shuttle PCR), followed by default melting curves.

In this study, total RNA was extracted from the different cultures of V. cholerae strains using an RNeasy kit (Qiagen, Beijing, China). A quantitative real-time reverse transcription PCR (qRT-PCR) was conducted using a One-Step SYBR Primerscript RT-PCR Kit II (TaKaRa, Beijing, China). Relative expression values were calculated as 2^{−(ΔCt target − ΔCt reference)}, Ct is the fractional threshold cycle, and the mRNA of recA was used as a reference for calculating the relative expression values. The following primer combinations were used: RecA-F (5′-GTGCTGTGGATGTCATCGTTGTTG-3′) and RecA-R (5′-CCACCACTTCTTCGCCTTCTTTGA-3′) for recA mRNA; tolC-F (5′-GGCACACTAAGCTCTGCCG-3′) and tolC-R (5′-CCGTATCTAAGCTTACCC-3′) for the mRNA of tolC; VC0229-F (5′-GCGATCACCTTTGTTGTAC-3′) and VC0229-R (5′-GCATAAGGCGTTTTTACCGC-3′) for the mRNA of the VC0229 gene; and VC0231-F (5′-GGTGCCTATGTTGGCCAAG-3′) and VC0231-R (5′-GATGTGCAGTGGTGCATTG-3′) for the mRNA of the VC0231 gene. A control mixture lacking reverse transcriptase was used to ensure that the chromosomal DNA was not contaminated.