Iqtm5 multicolor real time pcr detection system

The cDNA was synthesized from 1 μg of total RNA with PrimeScript^TM RT reagent kit with gDNA Eraser (Takara, Japan). SYBR PrimeScript^TM RT-PCR Kit (Takara, Japan) and IQ^TM5 multicolor real-time PCR detection system (BIO-RAD, United States) were used and each experiment included three independent technical and biological replications. RT-qPCR was carried out for 40 cycles (94°C for 5 s, 60°C for 34 s) after an initial denaturation step (94°C for 30 s). RPL5 (encoding ribosome protein L5) was used as an internal reference gene (Wu et al., 2019a (link)). U6 snRNA was used as an internal reference for miRNAs analysis (Wu et al., 2019b (link)). The data were calculated by 2^–ΔΔCt method. The primers were designed with Beacon Designer 7.7 and listed in Supplementary Table S1.

Total RNA from tissues and the cultured cells was extracted with the TRIzol reagent (Invitrogen, USA). Reverse transcription and qRT-PCR reactions were performed using a qSYBR-Green-containing PCR kit (Qiagen, USA) and a BioRad IQTM5 Multicolor Real-Time PCR Detection System (USA). The equation 2^−ΔΔCt was used to calculate the relative amount of miRNA among Which U6 snRNA was used as an internal control (Ct was the fractional cycle number at which the fluorescence of each sample passes the fixed threshold) and ΔΔCT = (C_{T miRNA} − C_{T U6})_target − (C_{T miRNA} − C_{T U6})_control. The primers of KRAS mRNA for qRT-PCR detection were as follows: forward, 5′-GGACTGGG-GAGGGCTTTCT-3′ and reverse, 5′-GCCTGTTTTGTGTCTACTGTTCT-3′. The primer was synthesized by Invitrogen. Each experiment was performed three times intriplicate.

The expression levels of ldt_Go and pbpb7_Go genes in exponential and stationary phase was analyzed by RT-PCR. RNA was isolated from G. oxydans wild-type cultures at OD₆₀₀ 0.3 units (Exp) or 3 units (Sta) using the RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions. Reverse transcription was performed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher), using aliquots of 5 µg total RNA. The synthesized cDNA was purified using a QIAquick PCR purification kit (Qiagen), and its concentration was determined spectrophotometrically in a Nano-drop Lite spectrophotometer (Thermo Scientific). Real time quantitative PCR (qRT-PCR) was performed using an iQ^TM5 Multicolor Real-Time PCR Detection System (BIO-RAD) with qPCRBIO SyGreen Mix fluorescein (PCR BIOSYSTEMS). Master mixes were prepared as recommended by the manufacturer, with qRT-PCR primers listed in Supplementary Table 4. Two independent experiments were carried out in triplicates for each data point. The relative quantification in gene expression was determined using the 2^−ΔΔCt method^{61 (link)}, using recA_Go(gox1522) gene as control.

RNA was extracted and purified from 81116 exposed to Fraquil or Brucella broth for 4 h at 4°C. Three biological replicates were tested. One μg of RNA was used for reverse transcription reactions along with a negative control without reverse transcriptase. qPCR was performed on an iQ^TM5 Multicolor Real-Time PCR Detection System (Bio-Rad) using iTaq universal SYBR green supermix (Bio-Rad) according to manufacturer’s protocol. Gene-specific primer sets were designed with the IDT primer design software (Bachman and Swanson, 2001 (link)) (Table 2) and their amplification efficiency was determined experimentally to be >85%. The 16S rRNA gene was used as a reference to normalize the data. Fold change was calculated as described previously (Livak and Schmittgen, 2001 (link)) and presented as a log2 ratio.

Total RNA from different organs of A. belladonna was extracted and reverse-transcribed to cDNA using the FastQuant RT Kit (TianGen Biotech, Beijing, China). Quantitative real-time PCR (qRT-PCR) was performed using the iTaq^® Universal SYBR^® Green Supermix (Bio-rad) and the IQTM5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and the 2^−ΔΔCT method was used to calculate the expression levels of the genes [25 (link)]. The phosphoglycerate kinase gene (PGK) was used as the internal reference gene [26 (link)]. Primers used for qRT-PCR are listed in Table S1. All experiments were carried out with three biological repeats.