The total RNA was extracted from samples treated with NaCl using HiPure HP Plant RNA Mini Kit (Magen, shanghai, China). Then, the RNA was used for cDNA synthesis with the PrimeScript™ RT reagent kit with gDNA Eraser (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was performed according to the supplier’s instructions of the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China) in the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR reaction system consists of one 5 µL cDNA sample, 0.6 µL primers, and 7.5 µL SYBR Green (Accurate Biotechnology, Hunan, China), 1.9 µL nuclease-free H2O, and the reaction volume was 15 µL. The PCR reaction was performed with the following conditions: 30 s at 95 °C, 40 cycles of 5 s at 95 °C, and 30 s at 60 °C. The grapevine β-actin (XM_034827164.1 ) were used as the internal references. All experiments were repeated at least three times and all the primers used in this study were listed in Table S1 .
Primescript rt reagent kit with gdna eraser
Manufactured by Vazyme
Sourced in China
The PrimeScript™ RT reagent kit with gDNA Eraser is a set of reagents designed for reverse transcription of RNA. It includes a gDNA Eraser component that eliminates genomic DNA contamination prior to reverse transcription.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq 2, by Vazyme (2 mentions)
Trizol reagent, by Vazyme (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hipure hp plant rna mini kit, by Magen Biotechnology Co (1 mentions)
Sybr green, by Accurate Biology (1 mentions)
Sybr green premix pro taq hs qpcr kit, by Accurate Biology (1 mentions)
7900ht qpcr system thermal cycler, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Abi 7500 rt pcr system, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq 2, by Thermo Fisher Scientific (1 mentions)
Tb green qpcr kit, by Takara Bio (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Chamq sybr qpcr master mix, by Vazyme (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
14 protocols using primescript rt reagent kit with gdna eraser
1
Quantitative Real-Time PCR for Grapevine Gene Expression
2
Quantitative RT-PCR for Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the cells and tissues using the Trizol reagent (Invitrogen). Approximately 1 μg of RNA was used for the reverse transcription reaction using the PrimeScript™ RT reagent Kit with gDNA Eraser (Vazyme). The cDNA was amplified with the following primers: 5′-CACAGACCTGGCCCGTTTT-3′ (forward) and 5′- AGTCCGGCCTTTCTTTTTGC-3′ (reverse) for ING4; 5′-CTTCCAAGTCTGGAGCGATGT-3′ (forward) and 5′-TACCGTCAAAGGGGTATCCAT-3′ (reverse) for MMP-2; 5′-GTACTCGACCTGTACCAGCG-3′ (forward) and 5′-AGAAGCCCCACTTCTTGTCG-3′ (reverse) for MMP-9; 5′-TCTCGACATCGAGGACCCAT-3′ (forward) and 5′-TGGACCAGTCGAAACCCTTG-3′ (reverse) for TIMP-2; 5′-CTGTCTAATGCCCTGGAGCC-3′ (forward) and 5′-ACGCGAGTCTGTGTTTTTGC-3′ (reverse) for VEGF; 5′-CTGGCGCTCAGCCATACAG′ (forward) and 5′-CGCACTTATACTGGTCAAATCCC′ (reverse) for COX-2; 5′-GGTGCCTTTTCACAGGCTC'-3′ (forward) and 5′-GCTGTTCTCATTGGGTGACTC-3′ (reverse) for Sp1; 5′-GCCGGTGCTGAGTATGTC-3′ (forward) and 5′-CTTCTGGGTGGCAGTGAT-3′ (reverse) for GAPDH.
Real time PCR was carried out in triplicate with SYBR Green PCR Master Mix using a 7900HT qPCR system thermal cycler (Applied Biosystems) as described previously [37 (link)]. GAPDH mRNA was used as an internal control for each sample, and the Ct value for each sample was normalized to GAPDH mRNA.
Real time PCR was carried out in triplicate with SYBR Green PCR Master Mix using a 7900HT qPCR system thermal cycler (Applied Biosystems) as described previously [37 (link)]. GAPDH mRNA was used as an internal control for each sample, and the Ct value for each sample was normalized to GAPDH mRNA.
3
Liver Total RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from liver tissue using Tissue Total RNA Isolation Kit V2 (cat. no. RC112; Vazyme Biotech Co., Ltd.) and was reverse-transcribed using PrimeScript™ RT Reagent Kit with gDNA Eraser (cat. no. RR047A; Takara Biotechnology Co., Ltd.). qPCR was performed using an ABI 7500 RT-PCR System (Applied Biosystems, Thermo, USA) and a TB Green® Premix Ex Taq™ II (cat. no. RR82LR; Takara Biotechnology Co., Ltd.). The sequence information of all primers used is listed in Table 1 . The following thermocycling conditions were used for the qPCR: initial denaturation for 30 s at 95°C followed by 40 PCR cycles (95°C for 5 sec, 60°C for 30 sec, and 72°C for 30 sec). The relative gene expression was calculated using the 2-ΔΔCq method [21 (link)] and normalized to the internal reference gene β-actin.
4
Colistin and SC Effects on E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Early‐exponential phase of E. coli G92 was exposed to colistin (0.5 µg mL−1) alone or in combination with SC (2 mg mL−1) for 4 h. After that, total RNA was extracted and measured using the 260/280 nm absorbance ratio. Using the PrimeScript RT reagent Kit with gDNA Eraser (Vazyme, Nanjing), reverse transcription of 1 µg of extracted RNA was carried out following the manufacturer's instructions. RT‐qPCR analysis was performed by 7500 Fast Real‐Time PCR System (Applied Biosystem, CA, USA) using the TB Green qPCR Kit (Takara) with the optimized primers (Table S3 , Supporting Information). The fold changes in mRNA expression relative to the reference genes (16S rRNA) in E. coli were calculated using a relative quantitative technique.
5
Quantitative RT-PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was reverse-transcribed to cDNA using the PrimeScript RT reagent kit with gDNA Eraser (Vazyme, China) according to the manufacturer’s instructions. The mRNA level of the target gene was quantified by RT-qPCR using ChamQ SYBR qPCR master mix (Vazyme). The reaction was performed using the ViiA-7 real-time PCR system (Applied Biosystems, USA) under the following conditions: 5 min at 50°C and then 5 min at 95°C, followed by 40 cycles of 10 s at 95°C and 35 s at 60°C. The results were normalized to A. pleuropneumoniae 16S rRNA and analyzed via the 2−ΔΔCT method (21 (link)). Reverse transcription-PCR (RT-PCR) was carried out as described previously (22 (link)). The PCR products were electrophoresed and photographed using an ImageQuant LAS 4000 system (GE Healthcare, New Jersey, USA).
6
Inflammatory Pathway Induction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM, RPMI 1640, and Opti-MEM medium were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum, Hoechst 33342, propidiumiodide (PI), penicillin G sodium salt, lipopolysaccharide (LPS), Freund’s complete adjuvant, and chicken type II collagen were obtained from Merck (Darmstadt, Germany). Antibodies against NLRP3, GSDMD, caspase-1, NF-κB, and p-NF-κB were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Tubulin, STING, p-STING, F4/80, and γ-H2AX were obtained From Abcam (Cambridge, United Kingdom). Mouse IL-1β and IL-18 uncoated ELISA kits were purchased from Thermos Fisher Scientific (Waltham, MA, USA). Lactate dehydrogenase (LDH) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent, PrimeScript RT Reagent Kit with gDNA Eraser, Effectene Transfection Reagent, and SYBR Premix Ex TaqII were obtained from Vazyme (Nanjing, China). CCCP and JSH-23 were purchased from MCE (New Jersey, USA). Human or mouse PBMC separation medium were obtained from Solarbio (Beijing, China).
7
Quantitative and Semi-Quantitative RT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For quantitative and semi-quantitative RT-PCR, total RNA was extracted from rice samples with Trizol reagent (Vazyme), and 5 μg RNA was reverse-transcribed with the PrimeScript RT reagent kit with gDNA Eraser (Vazyme) according to the supplier’s manual. Real-time PCR was done with a real-time thermal cycling system (Bio-Rad). SYBR-Green was used to detect gene abundances. Each reaction was done with three biological replicates. Data were analyzed using Bio-Rad CFX Manager software. For evaluating UGT76C2 expression in transgenic rice, semi-quantitative RT-PCR was employed. The synthesized cDNA was diluted five times (1:5), and 2 μl was used for analyzing the transcript level. OsActin1 was used as an internal reference gene. PCR reactions were performed in 25 μl of total reaction volume, with 25 cycles for amplifying OsActin1 and 32 cycles for amplifying UGT76C2. The primers for the RT-PCR assay are included in Supplementary Table S1 .
8
Quantification of circSNRK, miR-103-3p, and SNRK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For quantification of circSNRK, miR-103-3p, or SNRK mRNA, PrimeScript™RT reagent Kit with gDNA Eraser (Vazyme biotech, Nanjing, China) was used to synthesize the first-strand cDNA. Stem-loop qRT-PCR was performed using a FastStart Essential DNA Green Master (Vazyme biotech, Nanjing, China). GAPDH was used to normalize the expression of circSNRK and SNRK mRNA. For quantification of miR-103-3p, Bulge-loopTM miRNA qRT-PCR Primer Sets (one RT primer and a pair of qPCR primers for each set) specific for miR-103-3p is designed by RiboBio (Guangzhou, China), cDNA was synthesized with a miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme biotech, China). AceQ qPCR SYBR Green Master Mix (Vazyme biotech, Nanjing, China) was then used for real-time PCR. U6 was used to normalize the cellular miR-103-3p expression. The formula was: relative gene expression = 2−ΔΔCt. The primers are listed in Supplementary Table 1 .
9
Extraction and Quantification of RNA from Grapevine and Nicotiana
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the leaves of grapevine rootstock ‘A35’ and N. benthamiana using the improved CTAB method and TRIzol reagent (Invitrogen, Carlsbad, CA, USA), respectively, as previously described [3 (link), 22 (link), 23 (link)]. Then, RNA was used for cDNA synthesis with the PrimeScript™ RT reagent kit with gDNA Eraser (Vazyme, Nanjing, China). Quantitative reverse transcription PCR (qRT-PCR) was performed according to the instructions of the SYBR® PrimeScript™ RT-PCR Kit (TaKaRa, Dalian, China) in the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The actin1 (AY680701) and actin7 (XM_034827164) were used as internal references in the VvASMT1 expression pattern in grapevine under NaCl and mannitol stresses, and actin (XM_033660572.1 ) and Tubulin (N181029A17) genes were used as internal references to determine ROS scavenging-related target genes and VvASMT1 expression in Nicotiana benthamiana qRT-PCR analyses. All experiments were repeated at least three times, and all the primers used in this study are listed in S1 Table in S1 File . The data were automatically analyzed using the CFX Manager software program (version 1.1).
10
RNA Extraction and RT-qPCR Analysis of E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA extraction was carried out as previously described.[62 ] Total RNA of E. coli B2 treated with DCA, CIP, or their combination was isolated using the EASYspin Plus kit (Aidlab, Beijing, China) and quantified by the ratio of absorbance (260 nm/280 nm) using a Nanodrop spectrophotometer (Thermo Scientific, MA, USA). The first‐strand cDNA from all bacterial cells was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (Vazyme, Nanjing, China) following the manufacturer's protocols. Thermal cycling was performed by a two‐step PCR amplification standard procedure with 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 34 s. RT‐qPCR test was performed using a 7500 Fast Real‐Time PCR System (Applied Biosystem, CA, USA). The fold changes of gene expression were determined using the 2−ΔΔCt method. Primer sequences used in this study are listed in Table S3 in the Supporting Information.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!