Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For mRNA quantification, cDNA was synthesized using iScript RT Supermix (Bio-Rad), following the manufacturer's protocol. Quantitative real-time PCR (qRT-PCR) analysis was performed in triplicate using iQ SYBR green Supermix (BioRad) on an iCycler Real-Time Detection System (Eppendorf). The mRNA level was normalized to GAPDH or 18S as a housekeeping gene. The human primer sequences used are listed in the Supplementary Table S1 . For miRNA quantification, total RNA was reverse transcribed using the miScript II RT kit (Qiagen). Primers specific for human and mouse mmu-miR-33 (Qiagen) were used and values normalized to SNORD68 (Qiagen) as a housekeeping gene. Mmu-miR-33 quantification and quantitative real-time PCR were performed in triplicate using SYBR Green Master Mix (SA Biosciences) on an iCycler Real-Time Detection System (Eppendorf).
Icycler real time detection system
Manufactured by Eppendorf
The iCycler Real-Time Detection System is a thermal cycler designed for real-time PCR applications. It provides precise temperature control and optical detection capabilities to enable quantitative analysis of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Iscript rt supermix, by Bio-Rad (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr green master mix, by Qiagen (1 mentions)
Snord68, by Qiagen (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Evagreen supermix, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
3 protocols using icycler real time detection system
1
Quantitative Analysis of mRNA and miRNA Expression
2
Quantitative RT-PCR Analysis of mRNA Expression
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Total RNA was isolated from tissue using TRIzol reagent (Invitrogen) following manufacturer's protocol. For analyzing mRNA expression, cDNA was synthesized from mRNAs using iScript RT Supermix (Bio-Rad), according to manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed in duplicate using EvaGreen Supermix (BioRad) on an iCycler Real-Time Detection System (Eppendorf). The mRNA levels were normalized to 18S. The sequences of all the primers used for qRT-PCR are provided in Table 1 .
3
Quantifying mRNA Expression from Tissue
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Total RNA from tissue was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. For mRNA expression analysis, cDNA was synthesized using iScript RT Supermix (Bio-Rad), following the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) analysis was performed in duplicate using SsoFast EvaGreen Supermix (Bio-Rad) on an iCycler Real-Time Detection System (Eppendorf). The mRNA levels were normalized to 18S.
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