Total RNA was extracted from Vero E6 cells using the RNAsimple Total RNA kit (KR118, Tiangen Biotech, China). The cDNA synthesis was performed with random primers using the FastKing gDNA Dispelling RT SuperMix kit (KR116, Tiangen Biotech). Quantitative reverse transcription PCR (qRT-PCR) was performed as described previously [38 (link)] using SYBR Green I fluorescent dye (E168, Novoprotein, Suzhou, China) and a QuantStudio three real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The cDNA was generated by qRT-PCR using specific primers (PEDV ORF3-F: 5′-GCA CTT ATT GGC AGG CTT TGT-3′; PEDV ORF3-R: 5′-CCA TTG AGA AAA GAA AGT GTC GTA G-3′; β-actin-F: 5′-AGG CTC TCT TCC AAC CTT CCT T-3′; β-actin-R: 5′-ACG TCG CAC TTC ATG ATC GA-3′). The qRT-PCR volume was 25 µl, which consisted of 12.5 µl 2×SYBR Premix Ex Taq (TaKaRa Bio, Kusatsu, Japan), 0.5 µl (10 pmol l−1) of the forward primer, 0.5 µl (10 pmol l−1) reverse primer, 4 µl template DNA, and 7.5 µl sterile water. The reaction was conducted using a protocol consisting of 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a 5 s annealing period at 92 °C and a 1 min annealing period at 60 °C. The reaction was then subjected to an extended period at 95 °C and a cooling period at 50 °C for 30 s. Finally, the Ct values were recorded and analysed.
Fastking gdna dispelling rt supermix kit
Manufactured by Tiangen Biotech
Sourced in China
The FastKing gDNA Dispelling RT SuperMix kit is a reagent designed for the reverse transcription of RNA samples. It includes a reverse transcriptase enzyme, buffer, and other necessary components for the conversion of RNA to complementary DNA (cDNA).
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Rnasimple total rna kit, by Tiangen Biotech (9 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (6 mentions)
Superreal premix plus sybr green kit, by Tiangen Biotech (6 mentions)
Mircute plus mirna qpcr kit, by Tiangen Biotech (4 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (4 mentions)
Sybr premix ex taq, by Takara Bio (4 mentions)
Nanodrop one, by Thermo Fisher Scientific (4 mentions)
Ultra sybr mixture with rox, by CWBIO (3 mentions)
Cfx384 real time pcr system, by Bio-Rad (3 mentions)
Eastep super total rna extraction kit, by Promega (3 mentions)
Genered, by Tiangen Biotech (3 mentions)
Mircute plus mirna first stand cdna kit, by Tiangen Biotech (3 mentions)
Superreal premix plus, by Tiangen Biotech (3 mentions)
Mircute plus mirna first strand cdna kit, by Tiangen Biotech (3 mentions)
Mircute plus mirna qpcr kit sybr green, by Tiangen Biotech (3 mentions)
Primer premier 5, by Premier Biosoft (2 mentions)
Rnaprep pure plant plus kit, by Tiangen Biotech (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Superreal premix plus kit, by Tiangen Biotech (2 mentions)
70 protocols using fastking gdna dispelling rt supermix kit
1
Quantitative PEDV RNA Detection in Vero E6 Cells
2
Pm4 Gene Allele Identification
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Total RNA was isolated from the young leaf tissues using TRIzol reagent (Invitrogen, United States) following product instructions. Residual DNA was then removed, and the corresponding cDNA was synthesized using the FastKing gDNA Dispelling RT SuperMix kit (Tiangen) thus: a 10-µL reaction mixture containing 50 ng–2 µg RNA, 2 µL 5× gDNA buffer and ddH2O to 10 μL, 42°C for 3 min, then adding 2 µL 10× King RT buffer, 1 µL FastKing RT Enzyme Mix, 2 µL FQ-RT Primer Mix and ddH2O to 10 μL, 42°C for 15 min, 95°C for 3 min. The Pm4 alleles were first cloned using primers GH398/GH399, GH400/GH401, GH398/GH407, and GH407/GH400 by nested PCR (Supplementary Table S2 ) and were then sequenced by Sanger sequencing (Sangon Biotech) and compared to the reported Pm4a-4e (GenBank, Pm4b_V1: MT783929, Pm4b_V2: MT783930) alleles with Seqman software (DNASTAR, Madison, American). For the new haplotypes, the DNA sequences were translated to amino acids with Editseq software (DNASTAR, Madison, American) for further analysis. The cDNA sequences of cloned disease-resistant genes were taken from the National Centre for Biotechnology Information (NCBI) database and were used for analysis by constructing the phylogenetic tree using the neighbor-joining method with the Poisson model in the MEGA7 software (Kumar et al., 2016 (link)).
3
Gene Expression Analysis Protocol
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For the expression analysis, 1 µg RNA was used for reverse transcription. The cDNA was synthesized using a FastKing gDNA Dispelling RT SuperMix kit (TIANGEN) in accordance with the manufacturer’s instructions. qRT-PCR was performed using the UltraSYBR Mixture (with ROX; CWBio, Beijing, China) and the CFX96 real-time PCR detection system (Bio-Rad). The expression levels of detected genes were normalized to TUB2 expression. Error bars denote standard deviations of three biological replicates [18 (link)]. The primers used for the expression analysis are listed in Additional file 1 .
4
Helicobacter pylori Culture and Characterization
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Columbia blood agar base, brain heart infusion (BHI), Mueller–Hinton (MH) agar, and H. pylori selective supplement (Dent) SR0147E were obtained from Oxoid, United Kingdom. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), MEM nonessential amino acids, TRIzol reagent, and Live/Dead BacLight Bacterial Viability kits (Molecular Probes) were purchased from Thermo Fisher Scientific, United States. Sheep blood was procured from Pingrui Biotechnology, China. CLR was purchased from Dalian Meilun Biotechnology, China. MTZ and saponin were acquired from Sigma, Germany. Fastking gDNA Dispelling RT SuperMix Kit, Talent qPCR PreMix (SYBR Green) Kit, TIANamp Bacteria DNA kit, and 2 × Taq PCR Mix were purchased from TIANGEN, China. Anti-H. pylori antibody ab20459 was obtained from Abcam, United Kingdom. Alexa Fluor 488 and goat anti-rabbit were bought from Southern Biotech, United States.
5
Quantitative Gene Expression Analysis
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For expression analysis, 1 μg RNA was used for reverse transcription. The cDNA was synthesized by using FastKing gDNA Dispelling RT SuperMix kit (TIANGEN, Beijing, China) according to the manufacturers' instructions. qRT-PCR was performed by using UltraSYBR Mixture (with ROX; CWBio, Beijing, China) and the CFX96 real-time PCR detection system (Bio-Rad). Expression levels of detected genes were normalized to TUB2 expression. Error bars denote SD of three biological replicates [18] . The primers used for expression analysis are listed in Additional le 1.
6
Upland Cotton Transcriptional Analysis
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The upland cotton TM-1 materials of ovules and fibers at different developmental stages of −3, −1, 0, 1, and 3 dpa for ovules, and 5, 10, 15, 20, and 25 dpa for fibers were collected as the experimental samples for RT-qPCR detection analysis. The four-week-old upland cotton TM-1 leaves were sprayed with 100 μM IAA, GA, ABA, JA and 2 mM SA for 3, 6, and 9 h, respectively, with the untreated leaves as control. All the samples were immediately frozen in liquid nitrogen for further RNA extraction and cDNA synthesis. Total RNA of different materials was extracted using the RNAprep Pure Plant Plus Kit (TianGen, Beijing, China) according to the protocol guidelines. cDNA was obtained by RNA reverse transcription using FastKing gDNA Dispelling RT SuperMix kit (TianGen, Beijing, China) and was then used as templates for RT-qPCR assay with the designed gene specific primers by Primer Premier 5 software (PREMIER Biosoft, San Francisco, USA), and GhUBQ7 (DQ116441.1) was used as internal reference for normalization (Supplementary Table S24 ). A total of 20 μL final volume was mixed for each reaction that was launched in Light Cycler@480 (Roche, Basel, Switzerland).
7
Quantifying NLRP3, ASC, and Caspase-1 Expression
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Total RNA was extracted from the hippocampal tissues using an RNA simple Total RNA kit (Tiangen Biotech Co., Ltd.), and the FastKing gDNA Dispelling RT SuperMix kit (Tiangen Biotech Co., Ltd.) was used to synthesize cDNA. RT-qPCR was performed using the FastFire qPCR PreMix (SYBR-Green; Tiangen Biotech Co., Ltd.) according the manufacturer's protocol. The following primers were used for qPCR: NLRP3 forward, 5′-GGAGGAA GAAGAAGAGAGGAGAGGAG-3′ and reverse, 5′-CTTGAGA AGAGACCACGGCAGAAG-3′; ASC forward, 5′-ACAATGA CTGTGCTTAGAGACA-3 and reverse, 5′-CACAGCTCCAGA CTCTTCTTTA-3′; caspase-1 p20 forward, 5′-TGAATACAAC CACTCGTACACGTCTTG-3′ and reverse, 5′-CCAGATCCT CCAGCAGCAACTTC-3′; and GAPDH (mouse endogenous reference gene primers; cat. no. B661304). All primers were purchased from Shenggong Bioengineering Co., Ltd. The reaction conditions were in accordance with the manufacturer's instructions: Denaturation at 95° for 15 min, followed by 40 cycles of denaturation at 95° for 10 sec, annealing at 61.5° for 20 sec and extension at 72° for 30 sec; iQ™5 software (Bio-Rad Laboratories, Inc.) was used to conduct PCR amplification. The RNA expression levels were evaluated using the 2−ΔΔCq method (35 (link)). PRISM 8 software (GraphPad Software, Inc.) was used to evaluate the relative RNA level. All experiments were carried out >3 times.
8
RT-qPCR Quantification of mRNA Expression
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The total RNA was extracted using TRIzol reagent. The target cDNA was obtained by reverse transcription using a FastKing gDNA Dispelling RT SuperMix kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. A SuperReal PreMix Plus kit (TIANGEN, Beijing, China) was used to perform RT-PCR with specific primers as previously described.7 (link),9 (link) Primers for RT-PCR were synthesized by TaKaRa Biotechnology (Dalian, China). Each sample was tested on triplicates, and the mRNA expression was measured by the threshold cycle (CT) values. The relative expression of mRNA was calculated by the 2ΔΔCT method, and β-actin was used as an internal reference for mRNAs.
9
Quantification of Gene Expression by qRT-PCR
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Total RNA of samples was isolated by using RNAprep Pure Plant kit (Polysaccharides and Polyphenolics-rich) (Tiangen, Beijing, China) and then used to synthesize the first-strand cDNA with FastKing gDNA Dispelling RT SuperMix kit (Tiangen, Beijing, China). Primer pairs specific to tested genes were designed using the GenScript online tool7 and then used to quantify the expression level by quantitative real-time PCR (qRT-PCR) amplification (Supplementary Table 1 ). qRT-PCR was performed using a SYBR® Premix Ex Taq™ kit (TaKaRa) on an ABI prism 7,900 Real-Time PCR System. The sweetpotato Tublin gene was used as a reference and relative changes of gene expression was analyzed by the 2–ΔΔCT method (Livak and Schmittgen, 2001 (link)).
10
Quantification of Cardiac Allograft Vasculopathy Cytokines
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The aorta allografts were harvested at POD 40 for detection of the key cytokines in the process of CAV. Total RNA was extracted using an RNAprep Pure Tissue Kit (DP 431, TIANGEN BIOTECH, Beijing, China, http://www.tiangen.com ). The concentration and purity of the extracted RNAs were measured using a UV spectrophotometer. Total RNA was reverse transcribed into cDNA for expression analysis by using the FastKing gDNA Dispelling RT supermix kit (KR118, TIANGEN BIOTECH, Beijing, China). RT-PCR was performed using the 2×SYBR Green qPCR Master Mix (B21203, TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s protocol. Primer sequences used in this experiment are listed in Table 1
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