Transscript 2 all in one first strand cdna synthesis supermix

T. pyogenes ATCC19411, BM07-1, and BMH06-3 were cultured on blood plates for 36 h and suspended in NB medium (Solarbio) containing 8% FBS to the logarithmic phase. The T. pyogenes suspension was diluted to 1 × 10⁵ CFU/mL and cultured with luteolin (final concentrations: 1/8 MIC, 1/4 MIC, and 1/2 MIC). The bacterial dilutions were mixed with the same final concentration of DMSO as a control. Fifty milliliters of bacterial suspension were incubated at 37 °C for 36 h, and bacterial cells were collected by centrifugation at 10,000× g and 4 °C. Total RNA of bacterial cells was extracted by TRIzol Reagent (Ambion, Carlsbad, CA, USA), and cDNA was reverse-transcribed using TransScript^®II All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Then, the transcription level of tatD genes was measured using quantitative Real-Time PCR (qPCR), as described by Guo et al. [24 (link)]. The relative expression quantities of tatD genes were normalized against 16S rRNA, and primer sequences are shown in Table 1. The optimum concentration of luteolin for treating T. pyogenes (ATCC 19411, BM07-1 and BMH06-3) was determined and the other T. pyogenes strains were treated with that concentration of luteolin. Finally, the expression of tatD genes was examined using qPCR in the same manner.

Total RNA was extracted from all 16 testis samples of two age groups using Trizol reagent (TransGen, Beijing, China). The first-strand cDNA for mRNAs was synthesized from 500 ng of each total RNA sample using a TransScript II All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) following the manufacturer′s recommendations. cDNA synthesis for miRNAs was performed from 500 ng of each total RNA sample using a Mir-X™ miRNA FirstStrand Synthesis Kit (Takara, Shiga, Japan) according to the kit instructions. The qPCR was carried out using a TB Green™ Fast qPCR Mix (Takara, Shiga, Japan) on a LightCycler 96 Real-Time System (Roche, Switzerland). The specific primers used in qPCR were designed and synthesized by the Qingke Biological Company (Xi'an, China). Primer sequences are provided in Supplementary Table 1. Eight independent biological replicates were included in qPCR analysis. β-actin and U6 were used as internal control genes for expression normalization of mRNAs and miRNAs, respectively. The relative expression levels of mRNAs and miRNAs were calculated using the 2^−ΔΔCt method (24 (link)).

Total RNA was extracted from the leaves of 2-year-old I. asprella using a HiPure Plant RNA Mini Kit (Magen, Guangzhou, China). The polyadenylated RNA was reverse transcribed into cDNA using a TransScript II All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. Using cDNA as the template, the coding regions of IaAO1 and IaAU1 were amplified using the Primer STAR high-fidelity DNA polymerase (Takara, Dalian, China). The PCR products obtained were purified and ligated into the vector pEASY-T5 and subsequently recombinant plasmids were used to transform E. coli Trans1-T1 competent cells using a pEASY-T5 Zero Cloning Kit (TransGen Biotech, Beijing, China). Both of the recombinant plasmids were verified by sequencing. All the sequences of the primers used in this study are shown in Supplementary Table S1. Detail information about strains and plasmids is listed in Supplementary Table S2.