Total RNA was extracted from T and SEZ tissues using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA was treated with DNase (Qiagen) and cDNA was synthesized from 5μg of total RNA using Random Primers (Invitrogen) and a Superscript III First-Strand Synthesis System for real-time PCR (RT–PCR) (Invitrogen). The RT–PCR for Nestin, Gfap, Sox2 and MIB1 transcripts was performed using CFX96 RT–PCR (Biorad), RT2 qPCR Primer Assay and SYBR Green Master Mix (Qiagen) according to the manufacturer’s instructions. 18S was used as the housekeeping reference. Relative expression quantification was performed by the ΔΔCT method. Experiments were performed in triplicate and each experiment was repeated 3 times.
Superscript 3 first strand synthesis system for real time pcr rt pcr
Manufactured by Thermo Fisher Scientific
The Superscript III First-Strand Synthesis System for real-time PCR (RT–PCR) is a reverse transcription kit used to generate first-strand cDNA from RNA samples. The system includes the Superscript III reverse transcriptase enzyme, random hexamers, and other necessary reagents for the reverse transcription reaction.
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Lab products found in correlation
Random primer, by Thermo Fisher Scientific (1 mentions)
Rt2 qpcr primer assay, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Qiagen (1 mentions)
Dnase, by Qiagen (1 mentions)
Cfx96 rt pcr, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Bio-Rad (1 mentions)
Qpcr machine, by Bio-Rad (1 mentions)
2 protocols using superscript 3 first strand synthesis system for real time pcr rt pcr
1
Quantification of Neural Stem Cell Markers
2
Gene Expression Analysis by qPCR
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Total RNA of tissues was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA was treated with DNase and run on the gels to validate the purity and quality. The cDNA was synthesized from 1 μg RNA using the SuperScript III First-Strand Synthesis System for real-time PCR (RT-PCR) (Invitrogen). Real-time PCR was performed on a Bio-Rad qPCR machine using the SYBR Green PCR Master Mix, according to the manufacturer’s recommended procedures. The primers were as follows: GHS-R forward primer 5′-GGACCAGAACCACAAACAGACA-3′, GHS-R reverse primer 5′-CAGCAGAGGATGAAAGCAAACA-3′; This primer set flanks the intron, which allows us to distinguish its expression from GHS-R 1b. UCP1 forward primer 5′-GTGAAGGTCAGAATGCAAGC-3′, UCP1 reverse primer 5′-AGGGCCCCCTTCATGAGGTC-3′. 18S and β-actin were used as housekeeping genes.
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