We extracted total RNA from cells with a Trizol reagent (Invitrogen, Thermo Fisher, Waltham, MA, USA) following the manufacturer’s instructions [29 (link)]. We used a Nanodrop spectrophotometer (EuroClone, Milan, Italy) to check RNA yield and purity, and MoloneyMurine Leukemia Virus Reverse Transcriptase (Sigma-Aldrich) to transcribe the total RNA into cDNA from each sample. Using SensiFAST SYBR Hi-Rox (Bioline, LABGENE SCIENTIFIC SAZI, Châtel-Saint-Denis, Switzerland), we performed Real-time PCR on a StepOne™ Real-Time PCR System (Thermo Fisher, Waltham, MA, USA). To quantify gene expression, we used the comparative Ct method (DDCt), and the relative quantification was estimated as 2-∆∆Ct. Melting curve analysis excluded the presence of non-specific amplification products. The forward and reverse primer sequences are shown in Table 1 .
Sensifast sybr hi rox
Manufactured by Meridian Bioscience
Sourced in United States, United Kingdom
The SensiFAST SYBR Hi-ROX is a real-time PCR master mix that contains SYBR Green I dye and ROX passive reference dye. It is designed for fast, highly sensitive, and reproducible quantification of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions)
Nanodrop spectrophotometer, by Euroclone (2 mentions)
Moloney murine leukemia virus reverse transcriptase, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Dyne first strand cdna synthesis kit, by Dyne Bio (2 mentions)
Tri reagent, by Molecular Research Center (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Steponeplus instrument, by Thermo Fisher Scientific (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tetro cdna synthesis kit, by Meridian Bioscience (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep plus kit, by Zymo Research (1 mentions)
Trisure, by Meridian Bioscience (1 mentions)
Steponeplustm real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Miscript primer assay, by Qiagen (1 mentions)
Mirnaeasy kit, by Qiagen (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
12 protocols using sensifast sybr hi rox
1
RNA Extraction and Real-Time PCR Analysis
2
Analyzing mTERT Expression in Mouse Brains
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Frozen mouse brains were ground with a customised tissue grinder in liquid nitrogen and stored at −80°C. RNA was isolated from 30mg of powder using RNeasy® Lipid Tissue Mini Kit (Qiagen) following the manufacturer's protocol. Reverse transcription was performed with 1μg of RNA and SuperScript® III Reverse Transcriptase (Invitrogen).
mTERT expression in the brain samples was analysed using Real-time Quantitative PCR and GAPDH was used as reference gene in the analysis using the SensiFAST™ SYBR® Hi-ROX (Bioline), on a Step One Plus instrument (Applied Biosystems) with the programme consisting of 95°C for 2 min, 50 cycles of 5 seconds at 95°C and 20 seconds at annealing temperature of each primer pair and a melt analysis step at gradually increased temperature from 68°C to 95°C. The expression of mTERT was normalised to the expres-sion of the housekeeping gene mGAPDH and the results were calculated and reported with the format of compara-tive threshold cycle (ΔΔCT). Sequence and annealing temperatures of primers used are detailed in Table2 .
mTERT expression in the brain samples was analysed using Real-time Quantitative PCR and GAPDH was used as reference gene in the analysis using the SensiFAST™ SYBR® Hi-ROX (Bioline), on a Step One Plus instrument (Applied Biosystems) with the programme consisting of 95°C for 2 min, 50 cycles of 5 seconds at 95°C and 20 seconds at annealing temperature of each primer pair and a melt analysis step at gradually increased temperature from 68°C to 95°C. The expression of mTERT was normalised to the expres-sion of the housekeeping gene mGAPDH and the results were calculated and reported with the format of compara-tive threshold cycle (ΔΔCT). Sequence and annealing temperatures of primers used are detailed in Table
3
RNA Isolation and qRT-PCR Analysis
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RNA was isolated from cells and tissues with TRIzol (Invitrogen) according to the manufacturer’s instructions and cDNA was synthesised with oligo(dT) primer and the Tetro cDNA Synthesis Kit (Bioline). The cDNA was amplified using SensiFAST SYBR Hi-ROX (Bioline) and analysed with a 384-well QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Oligonucleotide primers used for PCR are shown in Supplementary Table 3 .
4
Quantifying Gene Expression in Frozen Muscles
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Frozen muscles were weighed and ground in liquid nitrogen. RNA was isolated from 5 to 15 mg of sample according to the Qiagen lipid RNA isolation kit protocol. Reverse transcription was performed using Superscript III Reverse Transcriptase (Invitrogen). qPCR was performed using primers as described (Baker et al., 2016 ; Ishaq, Schroder, Edwards, von Zglinicki & Saretzki, 2018 ) and SyBr Green (SensiFAST SYBR Hi‐ROX, Bioline). 18S was used as housekeeping gene. Annealing temperatures for all primers were 60°C. Expression is shown as 2−ΔΔCt values.
5
Quantifying EGFR Gene Expression by RT-qPCR
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500 ng of the total RNA isolated by the Direct-zol RNA Miniprep plus kit (Zymo Research, Irvine, CA, USA) and was retrotranscribed with SuperScript™ II Reverse Transcriptase (Bioline, GC biotech, Waddinxveen, The Netherlands), according to standard procedures. One µL of cDNA was employed to quantify the transcripts by real time RT-PCR using the SYBR Green Supermix (SensiFast SYBR Hi-ROX, Bioline, GC biotech, Waddinxveen, The Netherlands) and gene-specific primers. Real-time RT-PCR was performed by using a StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative quantity (RQ) was calculated normalizing for GAPDH gene and using a CTR sample as calibrator. Mean values and standard errors of RQ were generated from three biological replicates. The following primer sequences were used: EGFR-FW: 5′-AGGAAGAAGCTTGCTGGTAGC-3′; EGFR-REV: 5′-CTCTGGAAGACTTGTGGCTTG-3′; GAPDH-FW: 5′-CACCATCTTCCAGGAGCGAG-3′; REV 5′-CCTTCTCCATGGTGGTGAAGAC-3′.
6
Quantitative Real-Time PCR Gene Expression Analysis
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Total RNA was extracted from cells by Trizol reagent (Invitrogen) according to the manufacturer’s instructions (33 (link)). RNA yield and purity were checked using a Nanodrop spectrophotometer (EuroClone), and total RNA from each sample was reverse transcribed into cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase (Sigma-Aldrich). Real-time PCR was performed on a StepOne™ Real-Time PCR System (Thermo Fisher) using SensiFAST SYBR Hi-Rox (Bioline). The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2−ΔΔCt. The presence of non-specific amplification products was excluded by melting curve analysis. Statistical analyses on real-time PCR data were performed using the Relative Expression Software Tool (REST) (34 (link)). The forward and reverse primer sequences were reported in Table 1
7
Gene Expression Quantification via SmartChip Real-Time PCR
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Gene expression was assessed using the SmartChip Real-Time PCR System (WaferGen BioSystems, Fremont, USA). Sample and assay mixes were prepared with SensiFAST™ SYBR Hi-ROX (Bioline, London, UK) in 384-well source plates using a Freedom Evo 150 robot (Tecan, Mannedorf, Switzerland). Assay and sample mixes were then automatically loaded into the nanowells of a MyDesign SmartChip with WaferGen’s MultiSample NanoDispenser using a ‘384 assays × 12 samples’ dispensing layout. The final reaction volume per nanowell was 100 nl, with an equivalent of 100 pg unamplified cDNA (total RNA equivalents) loaded per reaction. SmartChips were run in the SmartChip Cycler, and the cycling conditions were comprised of 3 minutes activation at 95°C, and 40 cycles of 30 seconds at 95°C and 60s at 60°C, followed by a dissociation curve analysis from 60°C to 95°C. Cq values, generated by the software from the SmartChip Cycler were used for down-stream data-analysis.
8
Quantitative RT-PCR Analysis of Gene Expression
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The total RNA was extracted from cells using TRIsure (Bioline, London, UK), and cDNA synthesis was performed using 2 µg of total RNA and Superscript II reverse transcriptase (Thermo). The reaction conditions involved incubation at 45 °C for 10 min, followed by 95 °C for 10 min, and then 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min. SYBR Green (SensiFAST SYBR Hi-ROX, Bioline, London, UK) was used to quantify the PCR product, and the StepOnePlusTM Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) was employed for detection. The primer sequences used are listed in Supplementary Table S2 . Relative mRNA expression levels were determined using the 2−ΔΔCT method, with normalization to the expression levels of the housekeeping gene GAPDH.
9
Quantifying mRNA Expression in Immune Cells
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Total RNA was extracted using TRI reagent (Molecular Research Center, USA) and cDNA was synthesized with the Dyne First Strand cDNA Synthesis Kit (Dyne Bio, South Korea) according to the protocol of the manufacturer. mRNA was quantified using the StepOnePlus™ Real-Time PCR systems (Applied Biosystems, USA) with SensiFAST SYBR Hi-ROX (Bioline, USA). mRNA levels were normalized to that of β-actin. The following primers were used: β-actin 5′-GGACTT CGAGCAAGAGATGG-3′ and 5′-TGTGTTGGGGTACAGG TCTTTG-3′; IL-17A 5′-CAACCGATCCACCTCACC TT-3′ and 5′-GGCACTTTGCCTCCCAGAT-3′; RORC 5′-AGTCGGAAGGCAAGATCAGA-3′ and 5′-CAAGAGAGGTTCTGG GCAAG-3′; IL-21 5′-TGTGAATGACTTGGACCCTGAA-3′ and 5′-AAACAG GAAATAGCTGACCACTCA-3′; IRF-4 5′-CCTGCAAGCTCTTTGACACA-3′ and 5′-GAGTCACCTGGAATCTTGGC-3′; and IL-23R 5′-AGGTACTGGCAGCCTTGGAGTT-3′ and 5′-CCCTGTAGAGATGGAAGCAACTG-3′.
10
Quantification of mRNA Levels
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Total RNA was extracted using TRI Reagent (Molecular Research Center, USA) and cDNA was synthesized with the Dyne First-Strand cDNA Synthesis Kit (Dyne Bio, South Korea) according to the manufacturer’s protocol. mRNAs were quantified using the StepOnePlus™ Real-Time PCR system (Applied Biosystems, USA) with SensiFAST SYBR Hi-ROX (Bioline, USA). mRNA levels were normalized to that of βactin. The following primers were used: β-actin, 5'–GGACTTCGAGCAAGAGATGG-3' and 5'-TGTGTTGGGGTACAGGTCTTTG-3'; SMILE, 5'–GACCTGCTGCAAAGGCTGTTA-3' and 5'-CTGGTTGTTGTCGTTACCGCT-3'; PRKAA, 5'–ACACCCAACCTCAGACAAGG-3' and 5'-ATCACGTGACGGTGGTTACA-3'; and FOXP3, 5'–CACTGCCCCTAGTCATGGT-3' and 5'-GGAGGAGTGCCTGTAAGTGG-3’.
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