Total RNA for the qRT-PCR was isolated using TRIzol (Invitrogen, USA). The complementary DNA (cDNA) was reverse transcribed using HiScript II Q RT SuperMix for qRT-PCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The analysis was performed using the AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The primer sequences are shown in the Supplementary Table 2 .
Hiscript 2 q rt supermix for qrt pcr
Manufactured by Vazyme
Sourced in China
HiScript® II Q RT SuperMix for qRT-PCR is a ready-to-use reagent for reverse transcription and real-time PCR amplification. It contains a thermostable reverse transcriptase and a high-performance DNA polymerase, optimized for sensitive and efficient quantitative real-time PCR.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Chamq universal sybr qrt pcr master mix, by Vazyme (2 mentions)
Hrp conjugated affinipure goat anti rabbit igg h l, by Proteintech (2 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (2 mentions)
β actin antibody, by Proteintech (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Iqtm 5 optical module real time pcr detection system, by Bio-Rad (1 mentions)
Chamq sybr qpcr master mix, by Vazyme (1 mentions)
Ultrasybr mixture, by CWBIO (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Pierce bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Exorneasy midi kit, by Qiagen (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iqtm 5 connect real time system, by Bio-Rad (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by CWBIO (1 mentions)
Protease inhibitor cocktail, by CWBIO (1 mentions)
10 protocols using hiscript 2 q rt supermix for qrt pcr
1
Quantitative RT-PCR Analysis of RNA
2
Quantification of mRNA Expression
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According to the manufacturer’s protocol, total RNA was extracted with TRIzol Reagent (Invitrogen, USA), and then was reverse-transcribed with HiScript II Q RT SuperMix for qRT-PCR (Vazyme, China) (3 (link)). An iQTM 5 Optical Module Real-Time PCR Detection System (Bio-Rad, USA) was used to perform qRT-PCR. According to the manufacturer’s instructions, the ChamQTM SYBR qPCR master mix (Vazyme biotech, China) was used to quantify mRNA. The primers used are listed in Table S1
3
Quantification of Phoxim Degradation Genes
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To confirm the results of RNA-Seq and quantify the transcription of key genes related to phoxim degradation of strain YP6, a qRT-PCR using Ultra SYBR Mixture (CW0957S, CWBIO) was performed and the primers used were listed in Table S6 . YP6 control (without phoxim) and treatment samples (containing an initial phoxim concentration of 50 mg L−1) were both cultured at 30 °C, 200 rpm. Samples were collected at 12, 24, 48, 72, and 96 h, respectively. Total RNA of each sample was extracted and purified as per the steps mentioned in the section “RNA-seq transcriptomic analysis”. Then, cDNA was synthesized from total RNA with random primers by using HiScript® II Q RT SuperMix for qRT-PCR (R222-01, Vazyme, Nanjing, China). Finally, the obtained cDNA was used as a template for qRT-PCR. The 16s rRNA gene was chosen as an internal control. All reactions were performed in three biological replicates, and the 2−△△CT method [30 (link)] was used to calculate the gene expression levels for strain YP6.
4
Microcystin-LR Effects on Colonic Cells
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MC-LR with a purity ≥ 95% was purchased from Alexis Corporation (Lausen, Switzerland). L. fermentum was acquired from Henan BeNa Culture collection (BNCC). Normal colonic epithelial NCM460 cells were purchased from Otwo Biotech Inc., (Shenzhen, China). RPMI 1640 medium was purchased from Sigma (Livonia, MI, USA). RIPA buffer, bovine serum albumin (BSA), and the Pierce bicinchoninic acid (BCA) protein assay kit were purchased from Thermo Scientific Pierce (Rockford, IL, USA). The polyvinylidene fluoride (PVDF) membrane was purchased from Merck Millipore Ltd., (Billerica, MA, USA). Trizol Reagent was purchased from TaKaRa LA Taq (Shiga, Japan). The ChamQ Universal SYBR qRT-PCR Master Mix and HiScript® II Q RT SuperMix for qRT-PCR were purchased from Vazyme (Nanjing, China). The IL-6, TNF-α, IL-1β, and IL-10 ELISA kits were purchased from Neobioscience (Shenzhen, China). CSF1R antibody and Rap1b antibody were purchased from Abcam (Cambridge, UK). β-actin antibody and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) were purchased from Proteintech (Wuhan, China).
5
Quantifying RNA Expression in Cells and Exosomes
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The total RNA of the control and treated cells or exosomes was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany), the total RNA of exosomes was isolated using the exoRNeasy Midi Kit (Qiagen, Hilden, Germany), and the cDNA was synthesized using HiScript II Q RT SuperMix for qRT‐PCR (R223‐01, Vazyme). Quantitative real‐time PCR for mRNA and miRNA expression analysis was carried out using QuantStudioTM 7 Flex (Thermo Fisher Scientific), and this was normalized using β‐actin (for mRNA) or RNU6 (for miRNA). The data were analysed using the delta Ct method. The primers used for the present study are listed in Table 1 .
6
Relative Gene Expression Analysis in Grape
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The relative expressions of the genes involved in the pathways for sugar unloading, anthocyanin biosynthesis, and GBV biosynthesis were determined by qRT-PCR. Total spine grape RNA was extracted using the extraction kit (Bioteke #RP3302, Beijing, China) [22 (link)]. First-strand cDNA was reverse-transcribed by Hiscript II Q RT SuperMix for qRT-PCR (Vazyme #R223-01). The qRT-PCR reaction was performed using an iQTM5 Connect Real-Time System (Bio-Rad, Hercules, CA, USA). The specific primers used in this study are shown in Table S1 (Supplementary Materials ), and VdGAPDH was used as the internal reference gene to analyze the gene expression levels. The 2−ΔΔCT method was used to analyze the qRT-PCR data. All samples were analyzed in triplicate.
7
Quantifying Gene Expression via qRT-PCR
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cDNA for qRT–PCR was synthesized by using Vazyme HiScript II Q RT SuperMix for qRT–PCR (China, Cat: R223-01). VvACTIN-7 (VIT_04s0044g00580) was used as reference. There were three biological replicates for each experiment, which comprised three technical replicates, respectively. The relative expression was computed by the Δ–Δ method [60 (link)]. All qRT–PCR primers are listed in Supplementary Data Table S1.
8
Quantitative PCR Analysis of lncRNAs and mRNAs
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Total RNA (1 μg) from each sample was reverse-transcribed to cDNA using HiScript® II Q RT SuperMix for qRT-PCR (Vazyme). Quantitative PCR was performed on a ROCHE LightCycler® 480 instrument (ROCHE, Basel, Switzerland) using AceQ qRT-PCR SYBR Green Master Mix (without ROX) (Vazyme). All the primers were designed with Primer Premier 5.0 (PREMIER Biosoft, Palo Alto, CA). The qRT-PCR primers used in this study are listed in supplementary Table S1 . The relative standard curve method (2 − ΔΔCt) was used to determine the relative lncRNAs and mRNAs expression, β-actin was used as the reference.
9
Quantitative Analysis of Protein Expression
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MC-LR with a purity of 95% was purchased from Alexis Corporation (Lausen, Switzerland). RIPA buffer, bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime (Shanghai, China). The polyvinylidene fluoride (PVDF) membrane was purchased from Merck Millipore Ltd., (Billerica, MA, USA). The phosphatase inhibitor cocktail and protease inhibitor cocktail were purchased from CWBIO (Beijing, China). Trizol Reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The HiScript® II Q RT SuperMix for qRT-PCR and ChamQ Universal SYBR qRT-PCR Master Mix were purchased from Vazyme (Nanjing, China). The ERK antibody, p-ERK antibody, Raf antibody, p-Raf antibody, ZO-1 antibody, Occludin antibody, Claudin1 antibody, β-actin antibody, HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) were purchased from Proteintech (Wuhan, China).
10
Transcriptional Response to Cold Stress
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Bacterial cells grown overnight in TSB medium at 37°C were subcultured 1:1000 in fresh TSB broth. Cells grown up to the exponential phase (OD600; 0.6) were exposed to 4°C for a series of times. Total RNA was extracted from bacterial cultures using the Bacteria Total RNA Isolation Kit (Sangon Biotech, China). The synthesis of complementary DNA (cDNA) was achieved using the HiScript II Q RT SuperMix for qRT-PCR (Vazyme, China). For quantitative RT-PCR, the reactions were performed using SYBR qPCR Mix (Vazyme, China) and run in the CFX96 Real-Time System (Bio-Red, United States). Data were normalized using the 16S rRNA as internal control, and calculated using the 2−ΔΔCt method.
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