Total RNA was extracted using the Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. The RNA concentrations were quantified with the QubitFluorometer. Reverse transcription PCR was carried out by the Thermo script RT system (Invitrogen, Shanghai, China) according to the manufacturer's protocol. qPCR was performed in triplicate in a total volume of 20 μl containing 10 μl SsoFastEvaGreensupermix (Bio-Rad) with SYBR Green. The reaction was run on a Mini OpticonTM Real-time PCR instrument. Melting curve analysis was done to confirm the specificity of different genes. Fold-changes of genes with different treatment were calculated by normalizing the Ct values to the GADPH internal control. Primers for Elp3 were 5'-TTTGTAAAATGCCACAGGAGC -3' and 5'-GGCTCCTCTTGTAGAACTGCC-3'. Primers for Elp4 were 5'-GTTAAAAAATGAGAAATGGCGG-3' and 5'-TTCCTCCTTAGTCGCTGCAT-3'. Primers for MMP-2 were 5'-GACAACGCCCCCATACCA G-3' and 5'-CACTCGCCCCGTGTGTTAGT-3'. Primers for MMP-9 were 5'-ACGCAGACATCGTCATCCAGT-3' and 5'-ACGCAGACATCGTCATCCAGT-3'. Primers for GAPDH were 5'-GACCTGACCTGCCGTCTA-3' and 5'-AGGAGTGGGTGTCGCTGT-3'.
Thermoscript rt system
Manufactured by Thermo Fisher Scientific
Sourced in China
The Thermoscript RT system is a laboratory instrument designed for reverse transcription, the process of converting RNA into complementary DNA (cDNA). It provides a reliable and efficient platform for researchers to perform this essential step in various molecular biology applications, such as gene expression analysis and cDNA library construction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Trizol reagent, by Merck Group (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr mix, by Thermo Fisher Scientific (1 mentions)
Sybr green 1 pcr master mix, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
11 protocols using thermoscript rt system
1
Quantitative RNA Expression Analysis
2
Extracting and Quantifying mRNA
3
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Shanghai, China). Thermoscript RT system (Invitrogen, Shanghai, China) was applied for the reaction of reverse-transcription. The real time PCR was performed in triplicate in a total volume of 20 μl containing 10 μl SsoFastEvaGreensupermix (Bio-Rad) with SYBR Green, 2 μl cDNA, 2 μl each of the primers, and 6 μl RNase-free water. The PCR program was 94°C for 2 min, followed by 40 cycles at 94°C for 15 s, 60°C for 15 s, and 72°C for 30 s. qRT-PCR assay was run on a Mini OpticonTM Real-time PCR instrument. Each reaction contains 3 technical replicates for qRT-PCR analysis. Primers for RMP were 5'- TCC GAA TAA ATA CTG GAA AG -3' and 5'-AAG GCT CTG TAA ATG TCT GC -3'. Primers for Bax were 5'- TTT TGC TTC AGG GTT TCA TC -3' and 5'- GAC ACT CGC TCA GCT TCT TG -3'. Primers for Bcl-2 were 5'- GGT GGG AGG GAG GAA GAA -3' and 5'- CGC AGA GGC ATC ACA TCG -3'. Primers for GAPDH were 5'-GAC CTG ACC TGC CGT CTA-3' and 5'- AGG AGT GGG TGT CGC TGT -3'. Primers for HBx were 5'-ACCGACCTTGAGGCCTACTT-3' and 5'-GCTTGGCAGAGGTGAAAAAG-3'.
4
Quantification of ABCB5 and GAPDH Expression
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Total RNA was extracted from tissues and cell using TRIzol reagent. Reverse transcription (RT) was performed using the ThermoScript RT System (Invitrogen). Hot start PCR conditions were set as follows: 45 s at 94°C, 30 s at 55°C, and 1 min at 72°C for 28–30 cycles (ABCB5) or 26 cycles [for glyceraldehyde 3-phosphate dehydrogenase (GAPDH)]. The primers used in the study were as follows: ABCB5, 5′-TGTTTTGTTCGGGACCACCA-3′ (sense) and 5′-TTTCTCCGCCAGCATTCCAT-3′ (antisense); GAPDH, 5′-TGCCTCCTGCACCACCAACT-3′ (sense) and 5′-CCCGTTCAGCTCAGGGATGA-3′ (antisense).
5
RNA Extraction and RT-PCR Analysis of Lung Cancer
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By using the TRIzol reagent (Invitrogen), total RNA was extracted from lung cancer tissues and cell lines. RT was performed by using the Thermoscript RT System (Invitrogen). PCR conditions were set as follows: 45 s at 94°C, 30 s at 55°C, and 1 min at 72°C for 28–30 cycles (for MAD2B) or 26 cycles [for glyceraldehyde 3-phosphate dehydrogenase (GAPDH)]. The following primers were used in the study: MAD2B, 5′-AGAGTTTCATCCCCAAAGACAA-3′ (sense) and 5′-AGTTCAGGCAGTAGGCAAAGTC-3′ (antisense); slug, 5′-TCATCTTTGGGGCGAGTGAG-3′ (sense) and 5′-TGCAGCTGCTTATGTTTGGC-3′ (antisense); GAPDH, 5′-TGCCTCCTGCACCACCAACT-3′ (sense) and 5′-CCCGTTCAGCTCAGGGATGA-3′ (antisense).
6
Quantitative RT-PCR Analysis of Gene Expression
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Cells were lysed in TRIzol (Invitrogen Life Technologies), and total RNA was converted into cDNA by ThermoScript RT system (Invitrogen Life Technologies). Quantitative PCR was done in the StepOne Plus machine using SYBR Green PCR mix (Applied Biosystems). The values of target genes were normalized to the values of two housekeeping genes, GAPDH and CAP-1, and expressed as relative copy number, as described previously [18] (link)–[20] (link).
7
Quantitative Gene Expression Analysis
8
Monocyte RNA Quantification by RT-qPCR
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Monocytes were lysed in TRIzol reagent and stored at -80°C prior to RNA isolation. Total RNA was isolated according to the manufacturer’s recommendations and 1–2 μg of total RNA was converted to the first strand cDNA by the Thermoscript RT System (Invitrogen, Life Technologies, Carlsbad, CA) using poly-dT primer. Gene expression was quantified by real time PCR with Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) in the StepOne Real Time PCR System (Applied Biosystems) and expressed in relative copy numbers (RCN) as we describe elsewhere [10 (link)]. Briefly, RCN = 2-ΔCt x 100, with ΔCt calculated by subtracting the average Ct of two housekeeping controls (CAP-1 and GAPDH) from the experimental sample Ct.
9
Breast Cancer REV7 Gene Expression
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By using the TRIzol reagent (Invitrogen), total RNA was extracted from breast cancer tissues and cell lines. Reverse transcription (RT) was performed by using the ThermoScript RT System (Invitrogen). Polymerase chain reaction (PCR) conditions were set as follows: 45 s at 94°C, 30 s at 55°C, and 1 min at 72°C for 28–30 cycles (for REV7) or 26 cycles [for glyceraldehyde 3-phosphate dehydrogenase (GAPDH)]. The primers used in the study were as follows: REV7, 5′-AGAGTTTCATCCCCAAAGACAA-3′ (sense) and 5′-AGTTCAGGCAGTAGGCAAAGTC-3′ (antisense); GAPDH, 5′-TGCCTCCTGCACCACCAACT-3′ (sense) and 5′-CCCGTTCAGCTCAGGGATGA-3′ (antisense).
10
Extracting and Quantifying mRNA
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Cells or tissues were lysed in Trizol reagents (Sigma, T9424), and mRNA was extracted following the standard protocols, and reversed transcribed using the Thermoscript RT system (Invitrogen, 11146-016) according to the manufacture’s instructions. qRT-PCR was performed using Real-Time System (Sybr-green, Applied Biosystems, A25741). Data were normalized to the internal control and presented as a relative expression level. All primers for qRT-PCR are described in Table S1 .
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