The strain was collected in mid-exponential growth phase, which were further washed thrice with PBS and resuspended into 1 mL TRIzol (TaKaRa). After transferred into tubes containing glass beads (MP Biomedicals), the resuspended cells were vortexed by FastPrep-24TM5 G (MP Biomedicals) for 5 min to lyse the cells. Total RNAs were isolated according to the manufacturer’s instructions. The HiScript II 1st strand cDNA synthesis kit with gDNA wiper (Vazyme) was used to synthesis cDNA. The ChamQ Universal SYBR qPCR mix (Vazyme) and the QuantStudio 6 Flex real-time PCR system (Thermo) were further used to validate the genes transcript concentration. The parC served as the reference gene. The relative fold changes in gene expression were analysed by the 2−ΔΔCT method. All primers are listed in Table S2. A total of three independent experiments were performed.
Chamq universal sybr qpcr mix
Manufactured by Vazyme
Sourced in United States, China
ChamQ Universal SYBR qPCR Mix is a ready-to-use qPCR reagent that enables fast and accurate quantification of target DNA sequences. It contains all the necessary components for real-time PCR amplification and detection using SYBR Green chemistry.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Glass beads, by MP Biomedicals (1 mentions)
Hiscript 2 1st strand cdna synthesis kit with gdna wiper, by Vazyme (1 mentions)
Trizol, by Takara Bio (1 mentions)
Fastprep 24tm 5g, by MP Biomedicals (1 mentions)
Plant rna extract kit, by Tiangen Biotech (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Rna isolation total rna extraction reagent, by Vazyme (1 mentions)
Myeloperoxidase assay kit, by Nanjing Jiancheng (1 mentions)
Superoxide dismutase sod assay kit, by Nanjing Jiancheng (1 mentions)
Cdna synthesis kit, by Vazyme (1 mentions)
4 protocols using chamq universal sybr qpcr mix
1
RNA Extraction and qPCR Analysis
2
Quantifying Gene Expression in Plants
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Total RNA was isolated using plant RNA Extract Kit (Tiangen Biotech, China). The first-strand cDNA was synthesized following the instruction of Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). qRT-PCR was performed using a CFX Connect Real-time system (Bio-Rad, USA) with ChamQ Universal SYBR qPCR Mix (Vazyme, China). The relative expression levels were calculated using the 2-ΔCT method. GAPDH (Morgante et al., 2011 (link)) and AtACTIN7 (AT5G09810) were chosen as reference genes to normalize the relative expression level of CTS (Araip.H6S1B) in peanut and Arabidopsis, respectively.
3
Analyzing Chicken Immune Response to LPS
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Lipopolysaccharide (LPS, O55:B5) was purchased from Enzymax (China agent, Borealis Bio, Beijing). RNA Preservation Solution (202108) was purchased from Guangzhou Jiajie Biotechnology Co. (Guangzhou, China). Chicken D-LA ELISA KIT (202210) and Chicken DAO ELISA KIT (202210) were purchased from Guangzhou Jiajie Biotechnology Co. (Guangzhou, China). Superoxide Dismutase (SOD) assay kit (20220317) and Myeloperoxidase assay kit (20220308) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RNA isolation Total RNA Extraction Reagent (017E2272CA) and Cham Q Universal SYBR qPCR Mix (027E2201CA) were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). NF-κB antibody (GR3309451-1) was purchased from Abcam Plc. (Shanghai, China). GAPDH antibody (GR3309451-1) was purchased from Proteintech Group, Inc. (Wuhan, China). A real-time fluorescence PCR system (qTOWER3G, Kepeng Scientific Instruments Co, Guangzhou, China) and a cDNA synthesis kit (R312-01/02. Vazyme Biotech Co., Ltd. Nanjing, China) were used. The primers were provided by Sangon Biotech Co., Ltd. (Shanghai, China).
4
Quantifying Corneal Gene Expression
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Total RNA was extracted from corneal stromal cells or mouse corneas using an isolation kit (Trans Biotech, Beijing, China). Complementary deoxyribonucleic acids (cDNAs) were generated using a cDNA Synthesis Kit (Vazyme, Nanjing, China). Real-time quantitative polymerase chain reaction (RT–PCR) was performed by ChamQ Universal SYBR qPCR Mix (Vazyme), with GAPDH as an internal reference. The primers used in this study are listed in Supplementary Table S1 . Finally, the relative mRNA expression levels were determined using the 2−∆∆CT method.
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