PCR was used to detect NLRP3, caspase-1, IL-1β and IL-1α in the cell lysates. Total RNA was extracted from cells using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and the RNA quality and concentration were measured using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Inc.). Approximately 1 µg of total RNA was reverse-transcribed into complementary DNA using a FastQuant cDNA Kit (Tiangen Biotech Co. Ltd.) following the manufacturer's protocol. Quantitative PCR (95°C for 15 min followed by 39 cycles of 95°C for 10 sec and 63.8°C for 30 sec, with a final extension step of 65°C for 5 sec.) was performed using a CFX96TM Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The change in gene expression was calculated by 2−ΔΔC (19 (link)). The primer sequences used were as follows: NLRP3 forward, 5′-TGGCTGTAACATTCGGAGATTG-3′ and reverse, 5′-GAAGTCACCGAGGGCGTTGT-3′; caspase-1 forward, 5′-GGAAACAAAAGTCGGCAGAG-3′ and reverse, 5′-ACGCTGTACCCCAGATTTTG-3′; IL-1β forward, 5′-GGCCCTAAACAGATGAAGTGCT-3′ and reverse, 5′-TGCCGCCATCCAGAGG-3′; IL-1α forward, 5′-CAGTGAAATTTGACATGGGTG-3′ and reverse, 5′-CAGGCATCTCCTTCAGCAG-3′; GADPH forward, 5′-CCACATCGCTCAGACACCAT-3′ and reverse, 5′-GGCAACAATATCCACTTTACCAGAGT-3′.
Fastquant cdna kit
Manufactured by Tiangen Biotech
Sourced in China
The FastQuant cDNA kit is a laboratory reagent designed for the reverse transcription of RNA to cDNA. It is a tool used in molecular biology and gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Mirna cdna synthesis kit, by CWBIO (1 mentions)
Mirna qpcr assay kit, by CWBIO (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Total rna extraction kit, by Omega Bio-Tek (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sybr green 1, by Vazyme (1 mentions)
Primer premier v6, by Premier Biosoft (1 mentions)
Tianamp virus dna rna kit, by Tiangen Biotech (1 mentions)
Sybr green pcr master mix, by Roche (1 mentions)
Mx3005p detection system, by Agilent Technologies (1 mentions)
Sybr green real master mix, by Tiangen Biotech (1 mentions)
Total rna kit, by Omega Bio-Tek (1 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
9 protocols using fastquant cdna kit
1
NLRP3, Caspase-1, and Cytokine Expression Analysis
2
Quantitative PCR Analysis of Inflammatory Markers
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Total RNA was extracted using the total RNA Extraction Kit (Omega Bio-Tek, Norcross, GA, USA). The cDNA was synthesized using a FastQuant cDNA kit (Tiangen Biotech, Beijing, China). The real-time PCR system contained the following: 10 μL of SYBR Green premixture (2×), 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 8.0 μL of water, and 1.0 μL of cDNA, for a total volume of 20 μL. The primers used were as follows: IL-6, forward: 5′-ATGAAGTTTCTCTCCGCAAGA-3′ and reverse: 5′-CTAGGTTTGCCGAGTAGACCT-3′; IL-1β, forward: 5′-GACAAGCAACGACAAAATCCC-3′ and reverse: 5′-GAAGACAAACCGCTTTTCCATC-3′; TNF-α, forward: 5′-TACTGAACTTCGGGGTGATCG-3′ and reverse: 5′-TCCGCTTGGTGGTTTGCTAC-3′; GAPDH, forward: 5′-ATGGTGAAGGTCGGTGTGAA-3′ and reverse: 5′-TTACTCCTTGGAGGCCATGTA-3′. For miRNA real-time PCR, total miRNA was extracted using a mirVana miRNA Isolation kit (Ambion). MiRNA cDNA was synthesized using an miRNA cDNA Synthesis Kit (CWBIO, Beijing, China), and miRNA real-time PCR was conducted using an miRNA qPCR Assay Kit (CWBIO, Beijing, China). The primers used were as follows: miR-26b, forward: 5′- GCCGCTTCAAGTAATTCAGG-3′ and reverse: 5′- TATGGTTTTGACGACTGTGTGAT-3′; U6, forward: 5′- CAGCACATATACTAAAATTGGAACG-3′ and reverse: 5′- ACGAATTTGCGTGTCATCC-3′. Cycle threshold (CT) values among the different samples were compared using the 2−ΔΔCt method relative to GAPDH.
3
Quantifying DHCR7 Expression in 24-Methylene-Cholesterol Strains
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Total RNA from the mother strain and the two engineered strains producing 24-methylene-cholesterol was isolated using a Qiagen RNeasy Mini Kit (Qiagen, Germantown, MD, USA), and then assayed according to the manufacturer’s instructions. The total RNA obtained was then used as a template. First-strand cDNA was synthesized using a FastQuant cDNA kit (TIANGEN, Beijing, China) and then used for expression analysis of the DHCR7 gene in the two 24-methylene-cholesterol-producing strains. The primers used for RT-qPCR are listed in the Supplementary Materials (Table S1) .
4
eQTL Analysis of BCL11A in Epilepsy
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We performed eQTL analysis using 16 brain tissues from patients with drug-resistant epilepsy to investigate whether identified SNPs regulate BCL11A expression in human brain tissues. Tissue RNA Kit (Omega, United States) and the FastQuant cDNA kit (Tiangen, China) are used for genomic RNA extraction and cDNA reverse transcription, respectively, and the SYBR®Green I (Vazyme, China) and the ABI QuantStudio 6 Flex™ (ABI, United States) analyzer are used for Quantitative PCR. We designed the primers with Primer Premier V6.0 (Premier Biosoft Inc., United States). Details on Quantitative PCR premiers are provided in Supplementary Table 2 . Functional effect of identified SNPs on BCL11A expression was further verified by data from the Genotype-Tissue Expression project (GTEx).2
5
Viral Nucleic Acid Extraction and cDNA Synthesis
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Viral DNA and Viral RNA were extracted from the samples using the TIANamp Virus DNA/RNA kit (Tiangen, Beijing, China) according to the manufacturer's instructions. Reverse transcription of 100 ng viral RNA sample was conducted using the FastQuant cDNA Kit (Tiangen) according to the manufacturer's instructions. The extracted DNA and synthesized cDNA were stored at -20°C.
6
Quantifying Volatile Compound Genes in Grafted Plants
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The total RNA of grafted fruit was extracted by rapid universal plant RNA isolation Kit (Huayueyang, China), and the cDNA was obtained by reverse transcription amplification of total RNA by FastQuant cDNA Kit (Tiangen Biotech, Beijing, China). SYBR Green PCR Master Mix (Roche Diagnostics, Basel, Switzerland) was used for quantitative real-time RT-PCR on Roche lightcycle 96 real-time PCR system (Applied Biosystems, Waltham, MA, USA). The gene specific primers of RT-PCR are shown in Table 1 . The β-actin was used as an internal reference control gene to standardize gene expression data. The 2−ΔΔCt method was used to calculate the relative gene expression of key regulatory enzymes of volatile substances in grafted plants.
7
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was extracted as described above, and cDNA was reverse-transcribed from total RNA using FastQuant cDNA kit (Tiangen) following the manufacture' manual. RT-qPCR was performed using Mx3005P detection system (Agilent) at 95°C for 2 min, and then 40 cycles at 95°C for 20 s followed by 58°C for 20 s and 68°C for 20 s with ribosomal protein L27 gene (rpl27) as the endogenous control. cDNA equivalent to 20 ng of total RNA, 0.5 µM primer pairs and SYBR Green Real Master Mix (Tiangen) were used in each reaction. The 2−ΔΔCt method was applied to analyze relative gene expression levels [20] . Primers used in RT-qPCR were given in Table S1 .
8
Liver RNA Extraction and qPCR Analysis
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The total RNA was isolated from liver tissues using a Total RNA Kit (Omega Bio-Tek, Norcross, GA, USA). The preparation of the cDNA was carried out with a FastQuant cDNA kit (Tiangen, Beijing, China). The real-time PCR was carried out with SuperReal PreMix Plus (SYBR Green) (Tiangen, Beijing, China) for the quantification of target gene expression and normalized to the expression of GAPDH using 2−ΔΔCt method.
9
Gene Expression Quantification by qPCR
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All tissue samples were collected immediately after the animals were sacrificed. The tissues were immediately snap frozen in liquid nitrogen and preserved in -80°C till use. The tissues were further grinded into powder with liquid nitrogen. Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacture’s procedure. A total of 500 ng RNA was reverse transcribed by using FastQuant cDNA Kit (Tiangen, China). Subsequently, quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) in an Applied Biosystems 7300 instrument. Expression data were normalized using GAPDH as an internal reference gene and the relative expression levels were evaluated using the delta-delta cycle threshold method (ΔΔCt). Primers for the quantitative PCR are list in the S1 Table .
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