Heart and kidney samples were collected from rats under urethane anesthesia and frozen at −80°C. Next, the samples were homogenized on BeadBug microtube homogenizer (Benchmark Scientific, Inc). Total RNA was isolated from the samples according to the TRI Reagent protocol. cDNA was transcribed from RNA samples according to the iScript Reverse Transcription Supermix #1708841 protocol (Bio-Rad). The qPCR mixes were prepared according to the Bio-Rad SsoAdvanced Universal SYBR Green Supermix protocol #1725271. Amplifications were performed on a Bio-Rad CFX Connect Real-Time System under standardized conditions using commercial assays. Data were analyzed using CFX Manager 3.0 software. The genes investigated in this study were: angiotensinogen (Atg, qRnoCED0051666), angiotensin II receptor type 1a (At1a, qRnoCID0052626), angiotensin II receptor type 1b (At1b, qRnoCED0005729), angiotensin II receptor type 2 (At2, qRnoCED0007551), transforming growth factor-beta (Tgf-b, qRnoCED0007638), renin (Rn, qRnoCID0008721), metalloproteinase inhibitor 2 (Timp2, qRnoCID0001559). Beta-actin was used as housekeeping gene (Actbl2, qRnoCED0018219).
Ssoadvanced universal sybr green supermix protocol
Manufactured by Bio-Rad
The SsoAdvanced Universal SYBR Green Supermix is a ready-to-use qPCR reaction mix that contains all the necessary components for real-time PCR amplification and detection using the SYBR Green I dye. It is designed for sensitive and specific quantification of target sequences.
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2 protocols using ssoadvanced universal sybr green supermix protocol
1
Quantitative PCR Analysis of Renin-Angiotensin System
2
Quantification of Flavin Monooxygenase Genes in Rat Tissues
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In short, about 20 mg of every tissue was homogenized on BeadBug™ microtube homogenizer (Benchmark Scientific, Inc.). Total RNA was isolated from samples according to TRI Reagent® protocol. cDNA was transcribed from RNA samples according to iScript™ Reverse Transcription Supermix protocol (Bio-Rad). The qPCR mixes were prepared according to the Bio-Rad SsoAdvanced™ universal SYBR® Green Supermix protocol. Amplifications were performed in a Bio-Rad CFX Connect Real-Time System under standardized conditions using commercial assays.
We used semi-quantitative analysis of PCR products to carry out with glyceraldehyde 3-phosphate dehydrogenase (PrimePCR™ SYBR® Green Assay: Gapdh, Rat, qRnoCID0057018, Bio-Rad), actin (PrimePCR™ SYBR® Green Assay: Actb, Rat , qRnoCID0056984, Bio-Rad), succinate dehydrogenase (PrimePCR™ SYBR® Green Assay: Sdha, Rat , qRnoCID0057011, Bio-Rad) as internal references.
Genes investigated in this study were flavin containing monooxygenase 1 (PrimePCR™ SYBR® Green Assay: FMO1, Rat , qRnoCID0008990, Bio-Rad), flavin containing monooxygenase 3 (PrimePCR™ SYBR® Green Assay: FMO3, Rat, qRnoCID0003196, Bio-Rad) and flavin containing monooxygenase 5 (PrimePCR™ SYBR® Green Assay: FMO5, Rat , qRnoCID0053250, Bio-Rad).
We used semi-quantitative analysis of PCR products to carry out with glyceraldehyde 3-phosphate dehydrogenase (PrimePCR™ SYBR® Green Assay: Gapdh, Rat, qRnoCID0057018, Bio-Rad), actin (
Genes investigated in this study were flavin containing monooxygenase 1 (
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