Total RNA was isolated with cells using a reagent, and cDNA was reversed as directed by the manufacturer. RT-qPCR was performed using SYBR Green PCR Kit and was run on QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). All primers were synthesized by Tianyi Huiyuan Biotech Co., Ltd. Total RNA was isolated with cells using a reagent, and cDNA was reversed as directed by the manufacturer. RT-qPCR was performed using SYBR Green PCR Kit and was run on QuantStudio 7 Real-Time PCR System (Thermo Fisher Scientific). β-actin was used to normalize the expression levels of target genes. All primers were synthesized by Tianyi Huiyuan Biotech Co., Ltd. The oligos used in the RT-qPCR assays were as follows: β-actin F 5’-ATCATTGCTCCTCCTGAGCG-3’, R 5’- AGCTCAGTAACAGTCCGCC-3’; Tnfa F 5’-GATCGGTCCCAACAAGGAGG-3’, R 5’-GCTTGGTGGTTTGCTACGAC-3’; Il1b F 5’-GACTTCACCATGGAACCCGT-3’, R 5’- CAGGGAGGGAAACACACGTT-3’; Il6 F 5’-ACAAGTCCGGAGAGGAGACT-3’, R 5’-ACAGTGCATCATCGCTGTTC-3’. RT-qPCR analysis was carried out with a cycle setup of 3minat 95°C, 40 cycles of 5sat 95°C and 20sat 60°C.
Sybr green pcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, Israel, Netherlands, Switzerland, Japan
The SYBR Green PCR Kit is a reagent kit designed for real-time polymerase chain reaction (PCR) analysis. The kit contains all the necessary components, including SYBR Green I dye, to perform quantitative PCR (qPCR) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (218 mentions)
Trizol, by Thermo Fisher Scientific (64 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (31 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (29 mentions)
Cx96 detection system, by Bio-Rad (19 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (19 mentions)
Rneasy mini kit, by Qiagen (17 mentions)
Primescript rt reagent kit, by Takara Bio (17 mentions)
Iscript cdna synthesis kit, by Bio-Rad (16 mentions)
Rneasy kit, by Qiagen (12 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (12 mentions)
Abi 7300, by Thermo Fisher Scientific (11 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (11 mentions)
Abi prism 7900 sequence detector, by Thermo Fisher Scientific (10 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (9 mentions)
Advantage rt for pcr kit, by Takara Bio (9 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (9 mentions)
Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (9 mentions)
Rna 6000 nano kit, by Agilent Technologies (9 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
541 protocols using sybr green pcr kit
1
qPCR Analysis of Inflammatory Markers
2
Quantitative Analysis of TCF7L2 Expression
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Total RNA was extracted from pancreatic tissues by using the SYBR Green PCR kit (F-415XL, Thermo, United States). RNA was reverse-transcribed using the protocol provided in the kit (K1622, Thermo, United States). The primer sequences are listed in Table 1 . The gene was amplified through RT-PCR method using the SYBRGreen PCR kit (Thermo, United States). GAPDH was used as the reference gene. Amplification was run for 40 cycles. Samples were denatured at 95°C, followed by annealing at 60°C. The mRNA expression of the TCF7L2 gene was quantitatively analyzed using Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Thermo, United States). Data were analyzed with 2-ΔΔ CT (Schmittgen and Livak, 2008 (link)).
3
Mitochondrial RNA Expression Analysis
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RNA was extracted from isolated mitochondria by the PureLink RNA mini kit (Thermo Fisher Scientific, USA). The quantity and quality of extracted RNA were analyzed by using a NanoDrop 2000 spectrophotometer. Extracted RNA was prepared to synthesize cDNA according to the QuantiNova Reverse Transcription Kit. The QuantiNova SYBR Green PCR Kit was used to determine mRNA expression on an Applied Biosystems PCR machine (95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 30 s, and a final extension step of 60 °C for 30 s). The sequences of the primers used in the qRT–PCR experiment were as follows:
Cyt-c forward primer, 5’-CTG GGTGACGAGTGAAACTG-3’; reverse primer, 5’-TGAGCACAACAGGAACTGGA-3’; primer length in bp, 104 bp;
GAPDH forward primer, 5’-GAAATCCCATCACCATCTTCCAGG-3’; reverse primer, 5’-GAGCCCCAGCCTTCTCCATG-3’; primer length in bp, 120 bp.
Cyt-c forward primer, 5’-CTG GGTGACGAGTGAAACTG-3’; reverse primer, 5’-TGAGCACAACAGGAACTGGA-3’; primer length in bp, 104 bp;
GAPDH forward primer, 5’-GAAATCCCATCACCATCTTCCAGG-3’; reverse primer, 5’-GAGCCCCAGCCTTCTCCATG-3’; primer length in bp, 120 bp.
4
Quantitative Real-Time PCR Transcriptome Analysis
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Total RNA was extracted with TRIzol reagent (Invitrogen). Reverse transcription was performed with a PrimeScript RT‐PCR kit (Takara Biotechnology). Real‐time PCR was performed with a standard SYBR Green PCR kit (Applied Biosystems, Thermo Fisher). The thermal conditions were set as follows: 60°C for 1 min, 95°C for 15 min, 45 cycles of 95°C for 15 s, and the optimised annealing temperature for 1 min. GAPDH was used as an internal control. All specific primers are listed in Table S1 .
5
Quantitative Real-Time PCR Analysis
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Following treatment, the total cellular RNA was extracted by TRIzol reagents21 (link). The qPCR procedures using a SYBR Green PCR kit (Applied Biosystems, Suzhou, China) under the ABI Prism7500 Fast Real-Time PCR system were reported before22 (link). To calculate the product melting temperature, the melting curve analysis was carried out. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was examined as the internal control and reference gene. Quantification was through the 2−∆∆Ct method. The mRNA primers for Nrf2, HO1, NQO1, GCLC, and GAPDH were provided by Dr. Di18 (link). The primers of immunoresponsive gene 1 (IRG1) were purchased from OriGene (Beijing, China).
6
Quantifying Gene Expression via RT-qPCR
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Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 µg of each sample was reverse-transcribed into cDNA with random primers using a Reverse Aid M-MuLV reverse transcription kit (Fermentas, Hanover, MD). The relative mRNA expression levels were determined using real-time PCR and gene-specific primers with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) with a SYBR Green PCR kit (Applied Biosystems). The primers used are shown in Supplementary Table 1 . The expression levels of each gene were normalized to those of Tbp1, a housekeeping gene.
7
Quantitative Real-Time PCR Analysis
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Cellular RNA was extracted via TRIzol reagents (Promega, Madison, WI). For quantitative real-time PCR, a SYBR Green PCR kit (Applied Biosystems, Foster City, CA) was utilized for reverse transcription under the ABI Prism7600 Fast Real-Time PCR system. The primers of human GCLC, Nrf2, HO1, NQO1, and GAPDH have been described previously [42 (link)]. Primers of murine GCLC, Nrf2, HO1, NQO1, and GAPDH have also been described [43 (link)].
8
Quantification of PAK1 Gene Expression
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The total RNA of each group was extracted using a Trizol reagent and quantified by a NanoDrop ND-1000 spectrophotometer. The first strand cDNA was synthesized by reverse transcription with PrimeScript RT Master Mix. Thereafter, a Takara SYBR Green PCR Kit was used and RT–qPCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems, California, USA). The relative mRNA abundance of the PAK1 gene was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed by the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primer sequences of the PAK1 are listed as follows: Forward, 5′-GCTACAGGTGAGAAAACTGAGGT-3′; Reverse, 5′-TTCAATGCTGGACACACGGT-3′.
9
RNA Extraction and qRT-PCR Analysis
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Total RNA was prepared using Total RNA Mini Kit (IBI Scientific, IA); the first cDNA strand was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA). Manufacturers’ protocols were followed in each case. The PCR primers for ATG3, 5, 7, 10, 12, HK2, G6PD, PFK1, PFKP, PDGM2, and β-actin are listed in Supplemental Table-1 . SYBR green PCR kit (Applied Biosystems) was used according to the manufacturer's instructions. AB7300 system was used as follows: activation at 95°C; 2 minutes, 40 cycles of denaturation at 95°C; 15 seconds and annealing/extension at 60°C; 60 seconds, followed by melt analysis ramping from 60°C to 95°C. Relative gene expression was determined by the ΔΔCt method using β-Actin to normalize.
10
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted from cells using RNAiso Plus (Takara Bio Inc., Kusatsu, Japan), and complementary DNA (cDNA) was synthesized with Moloney murine leukemia virus reverse transcriptase (Promega, Fitchburg, WI, USA) in accordance with the manufacturer’s protocol. The original amount of the specific transcripts was measured by real-time PCR with an SYBR Green PCR kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) using the ABI Prism 7900 sequence detector (Applied Biosystems). The expression of specific genes was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression. Primers used are listed in Additional file 2 : Table S1.
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