Total RNA was isolated from cells using TRIzol (Invitrogen) according to the manufacturer's protocol. RNA was reverse-transcribed using the RT2 First Strand Kit and MMLV-RT (Takara, Dalian, China). The cDNA was subjected to real-time PCR amplification using gene-specific primers and 2× Brilliant II SYBR Green QPCR Master Mix (Invitrogen, La Jolla, CA, USA) as described previously (Zhao et al. 2012 (link)). Primer sequences are listed in Table 1 . Specific gene expression was calculated using the comparative 2−ΔΔCt method with GAPDH as the calibrator.
Brilliant 2 sybr green qpcr master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
Brilliant II SYBR Green qPCR Master Mix is a reagent that enables quantitative real-time PCR (qPCR) analysis. It contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescent signal, allowing for the detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Mmlv rt, by Takara Bio (2 mentions)
Rt2 first strand kit, by Takara Bio (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Abi 7500 thermocycler, by Thermo Fisher Scientific (1 mentions)
Mx3000p qpcr system, by Agilent Technologies (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Abi 7900ht real time pcr system, by Thermo Fisher Scientific (1 mentions)
Improm 2 reverse transcription system, by Promega (1 mentions)
5 protocols using brilliant 2 sybr green qpcr master mix
1
Quantitative RT-PCR Gene Expression Analysis
2
Quantifying GPER Gene Expression in Cells
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Total RNA was isolated from cells using TRIzol (Invitrogen, La Jolla, CA, USA) according to the manufacturer's protocol. Reverse transcription was used the RT2 First Strand Kit and MMLV-RT (Takara, Dalian, China). The cDNA was subjected to real-time PCR amplification using gene specific primers and 2× Brilliant II SYBR Green QPCR Master Mix (Invitrogen) as described previously (Zhao et al. 2012) .
Specific primer pairs for GPER: forward primer 5'-TCTCTAACCTCCGCAACCAC-3' and reverse primer 5'-CTGGGGGTGGAGACAAGCAT-3'. Primer pairs for Actin: forward primer D r a f t 10 5'-ACTATCGGCAATGAGCGGTTCC-3' and reverse primer 5'-AGCACTGTGTTGGCA TAGAGGTC -3'. Real-time PCR was carried out on an ABI 7500 thermocycler (Applied Biosystems, CA, USA). Specific gene expression was calculated using the comparative 2 -∆∆Cт method with Actin as the calibrator.
Specific primer pairs for GPER: forward primer 5'-TCTCTAACCTCCGCAACCAC-3' and reverse primer 5'-CTGGGGGTGGAGACAAGCAT-3'. Primer pairs for Actin: forward primer D r a f t 10 5'-ACTATCGGCAATGAGCGGTTCC-3' and reverse primer 5'-AGCACTGTGTTGGCA TAGAGGTC -3'. Real-time PCR was carried out on an ABI 7500 thermocycler (Applied Biosystems, CA, USA). Specific gene expression was calculated using the comparative 2 -∆∆Cт method with Actin as the calibrator.
3
Analyzing Tendon Marker Expression in Chick TPCs
4
Quantifying miRNA and mRNA Expression
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Total RNA was extracted using TRIzol (Invitrogen) in accordance with manufacturer’s protocol. Reverse transcription of total miRNA was performed by using a miScript reverse transcription kit (Qiagen). MiScript SYBR Green PCR kit (Qiagen) together with a pair of miR-214 specific primers was used for mature miRNA detection. RNU6B was used as an internal normalized reference.
For detection of UCP2 mRNA expression, total RNA was reversely transcribed by using SuperScript III First-Strand Synthesis System (Invitrogen) according to manufacturer’s instruction. Real-time PCR was performed in triplicate in an ABI 7900HT real-time PCR system (Applied Biosystems) with a 20 μl reaction mixture containing Brilliant II SYBR Green qPCR master mix and 300 nM UCP2 primers. Primer sequences for UCP2 were 5’-cgcatcggcctgtatgattc-3’ and 5’- cataggtcaccagctcagc-3’. Primer sequences for GAPDH were 5’-gagtcaacggatttggtcgt-3’ and 5’-ttgattttggagggatctcg-3’. The thermal cycling was initiated by polymerase activation step for 10 min at 95 °C followed by 40 cycles of denaturation (95 °C for 30 s) and annealing/extension (60 °C for 1 min). Relative expression of UCP2 was normalized to GAPDH as an internal control and determined by a previously described method [25 (link)].
For detection of UCP2 mRNA expression, total RNA was reversely transcribed by using SuperScript III First-Strand Synthesis System (Invitrogen) according to manufacturer’s instruction. Real-time PCR was performed in triplicate in an ABI 7900HT real-time PCR system (Applied Biosystems) with a 20 μl reaction mixture containing Brilliant II SYBR Green qPCR master mix and 300 nM UCP2 primers. Primer sequences for UCP2 were 5’-cgcatcggcctgtatgattc-3’ and 5’- cataggtcaccagctcagc-3’. Primer sequences for GAPDH were 5’-gagtcaacggatttggtcgt-3’ and 5’-ttgattttggagggatctcg-3’. The thermal cycling was initiated by polymerase activation step for 10 min at 95 °C followed by 40 cycles of denaturation (95 °C for 30 s) and annealing/extension (60 °C for 1 min). Relative expression of UCP2 was normalized to GAPDH as an internal control and determined by a previously described method [25 (link)].
5
Quantitative RT-PCR Analysis of Brain Transcripts
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Total RNA was isolated from brain cortex using Trizol® (Life Technologies, Thermo Fisher). RNA concentrations were determined using a NanoDrop ND-certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted July 31, 2019. ; https://doi.org/10.1101/616144 doi: bioRxiv preprint 1000 instrument (Thermo Fisher). Reverse-transcriptase reactions were performed with the ImProm-II Reverse Transcription System (Promega, Madison, USA) using 2 μg total RNA. qPCR was performed using Brilliant® II SYBR® Green QPCR Master Mix (Applied Biosystems, Thermo Fisher) and the Mx3000P QPCR System; MxPro qPCR software was used for the analysis (Stratagene, San Diego, CA USA). Primers sequences are shown in Table 1.
For quantification, target genes were normalized using Beta-actin (Actb) as housekeeping gene. The threshold cycles (Ct) were determined for each sample. The relative expression of mRNA was calculated using the 2 -ΔΔCt method (Livak and Schmittgen 2001) expressed relative to those from the PBS group.
The copyright holder for this preprint (which was not this version posted July 31, 2019. ; https://doi.org/10.1101/616144 doi: bioRxiv preprint 1000 instrument (Thermo Fisher). Reverse-transcriptase reactions were performed with the ImProm-II Reverse Transcription System (Promega, Madison, USA) using 2 μg total RNA. qPCR was performed using Brilliant® II SYBR® Green QPCR Master Mix (Applied Biosystems, Thermo Fisher) and the Mx3000P QPCR System; MxPro qPCR software was used for the analysis (Stratagene, San Diego, CA USA). Primers sequences are shown in Table 1.
For quantification, target genes were normalized using Beta-actin (Actb) as housekeeping gene. The threshold cycles (Ct) were determined for each sample. The relative expression of mRNA was calculated using the 2 -ΔΔCt method (Livak and Schmittgen 2001) expressed relative to those from the PBS group.
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