The total RNA was extracted using Ultrapure RNA Kit according to the manufacturer’s instructions (CWBIO, Taizhou, China). The RNA was reverse transcribed into cDNA using a Superscript II reverse transcription kit (Vazyme, Nanjing, China). Then, we used a SYBR-Green master kit (Vazyme, Nanjing, China) on a quantitative real-time PCR instrument (Applied Biosystems, USA). GAPDH or U6 was used for standardization. The primers used to amplify all of mRNA and miRNA involved in this study were chemically synthesized by TSINGKE (TSINGKE, Beijing, China) and the primer sequences used for qRT-PCR are shown in Supplementary Table 3 .
Real time quantitative pcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
A real-time quantitative PCR (qPCR) instrument is a laboratory equipment used to amplify and quantify a targeted DNA or RNA sequence. It measures the accumulation of amplified product during the PCR process in real-time, allowing for precise quantification of the initial target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Rnasimple total rna kit, by Tiangen Biotech (2 mentions)
Maxima sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Sybr green master kit, by Vazyme (1 mentions)
Superscript 2 reverse transcription kit, by Vazyme (1 mentions)
Ultrapure rna kit, by CWBIO (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Sybr green mix, by Roche (1 mentions)
Reveraid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Iscript reverse transcriptase, by Bio-Rad (1 mentions)
Taqman gene expression assay id, by Thermo Fisher Scientific (1 mentions)
Rnazol, by Molecular Research Center (1 mentions)
5 protocols using real time quantitative pcr instrument
1
Quantitative Real-Time PCR for Gene Expression
2
Cytokine Expression in Blood Cells
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The peripheral blood of patients was taken before treatment and 14 days after treatment. After the erythrocytes were lysed, the RNA of white blood cells was extracted with an RNA extraction kit (Kangwei Co., Beijing, China), which was then transcribed to cDNA with a reverse transcription kit (TaKaRa, Dalian, China). Based on gene sequences provided by GenBank, primers for interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β) were designed with optimized annealing temperatures, and then quantitative real-time PCR was conducted with SYBR Green mix (Roche, Basel, Switzerland). Data were collected via a quantitative real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). The mRNA expression of IL-6, TNF-α, and IL-1β of each patient before and after treatment was analyzed and expressed as the mean ± SD.
3
Quantitative Analysis of Gene Expression
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Total RNA was extracted utilizing the RNAsimple Total RNA kit (Tiangen). The ReverAid FirstStrand cDNA Synthesis kit (Thermo Scientific) was used to synthesize cDNA. The mRNA expression level was analyzed by real-time quantitative PCR instrument (Applied Biosystems) with Maxima SYBR Green qPCR Master Mix (Thermo Scientific). The primers’ sequences were shown as follows: human AFP, 5′-GCAGTGAATCTACAGGGACGC-3′ and 5′-ATCCTGATCCAACCAATCACC-3′; human HBP1, 5′-TGAAGGCTGTGATAATGAGGAAGAT-3′ and 5′-CATAGAAAGGGTGGTCCAGCTTA-3′; and human GAPDH, 5′-CCATGGAGAAGGCTGGGG-3′ and 5′-CAAAGTTGTCATGGATGACC-3′; mouse HBP1, 5′-TGGGAAGTGAAGACAAAT-3′ and 5′-TGACAGGGAGGACATACA-3′; mouse AFP, 5′-AAACCTCCAGGCAACAACCA-3′ and 5′-ACTCCAGCGAGTTTCCTTGG-3′; mouse IL-1β, 5′-GAAATGCCACCTTTTGACAGTG-3′ and 5′-TGGATGCTCTCATCAGGACAG-3′; mouse TNF-α, 5′-CAGGCGGTGCCTATGTCTC-3′ and 5′-CGATCACCCCGAAGTTCAGTAG-3′; mouse type I collagen, 5′-GCTCCTCTTAGGGGCCACT-3′ and 5′-CCACGTCTCACCATTGGGG-3′; mouse type III collagen, 5′-ACGTAGATGAATTGGGATGCAG-3′ and 5′-GGGTTGGGGCAGTCTAGTG-3′; mouse β-actin, 5′-AGCCATGTACGTAGCCATCC-3′ and 5′-GCTGTGGTGGTGAAGCTGTA-3′.
4
Quantifying Zebrafish GAD1 Expression via qPCR
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted utilizing the RNAsimple Total RNA kit (Tiangen). The ReverAid FirstStrand cDNA Synthesis kit (Thermo Scienti c) was used to synthesize cDNA. The mRNA expression level was analyzed by real-time quantitative PCR instrument (Applied Biosystems) with Maxima SYBR Green qPCR Master Mix (Thermo Scienti c). The primers' sequences were shown as follows: human AFP, 5'-GCAGTGAATCTACAGGGACGC-3' and 5'-ATCCTGATCCAACCAATCACC-3'; human HBP1, 5'-TGAAGGCTGTGATAATGAGGAAGAT-3' and 5'-CATAGAAAGGGTGGTCCAGCTTA-3'; and human GAPDH, 5'-CCATGGAGAAGGCTGGGG-3' and 5'-CAAAGTTGTCATGGATGACC-3'; mouse HBP1, 5'-TGGGAAGTGAAGACAAAT-3' and 5'-TGACAGGGAGGACATACA-3'; mouse AFP, 5'-AAACCTCCAGGCAACAACCA-3' and 5'-ACTCCAGCGAGTTTCCTTGG-3'; mouse IL-1β, 5'-GAAATGCCACCTTTTGACAGTG-3' and 5'-TGGATGCTCTCATCAGGACAG-3'; mouse TNF-α, 5'-CAGGCGGTGCCTATGTCTC-3' and 5'-CGATCACCCCGAAGTTCAGTAG-3'; mouse type collagen, 5'-GCTCCTCTTAGGGGCCACT-3' and 5'-CCACGTCTCACCATTGGGG-3'; mouse type collagen, 5'-ACGTAGATGAATTGGGATGCAG-3' and 5'-GGGTTGGGGCAGTCTAGTG-3'; mouse β-actin, 5'-AGCCATGTACGTAGCCATCC-3' and 5'-GCTGTGGTGGTGAAGCTGTA-3'.
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