RNA was isolated from cells using Total RNA isolation kit (Norgen Biotek Corp, Thorold, Ontario, Canada) following the manufacturer’s instructions. Briefly, first-strand cDNA was prepared using 1–2ug of total RNA, the Superscript III RT kit and random hexamer primers (Invitrogen, Carlsbad, CA, USA) in a total volume of 25 µL. Reverse transcription reaction was carried out for 90 minutes at 50°C and an additional 10 minutes at 55°C. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on an ABI Prism 7900 sequence detection system using SybrGreen reagents (Takara Bio Company, Clontech, Mountain View, CA, USA) and TaqMan® Gene Expression Master Mix (Life Technologies, Madrid, Spain). Primers used were previously described.20 (link),22 (link),23 (link)
Sybrgreen reagents
Manufactured by Takara Bio
Sourced in United States
SybrGreen reagents are a type of fluorescent dye used in molecular biology applications, such as real-time PCR (qPCR) and other nucleic acid detection techniques. The reagents bind to double-stranded DNA, emitting a fluorescent signal that can be detected and quantified during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression master mix, by Thermo Fisher Scientific (4 mentions)
Superscript 3 rt kit, by Thermo Fisher Scientific (4 mentions)
Random hexamer primer, by Thermo Fisher Scientific (3 mentions)
Total rna isolation kit, by Norgen Biotek (3 mentions)
Abi prism 7900, by Takara Bio (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 flex, by Thermo Fisher Scientific (1 mentions)
Rna purification kit, by Norgen Biotek (1 mentions)
5 protocols using sybrgreen reagents
1
Quantitative RNA Expression Analysis
2
Quantitative Real-Time PCR Analysis
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Total RNA (1–2 μg) was reverse-transcribed with Superscript III RT kit according to the manufacturer's instructions. Real-time PCR (RT-PCR) was performed in an ABI Prism 7900 sequence detection system using SybrGreen reagents (Takara Bio Company) and TaqMan® Gene Expression Master Mix (Life Technologies). Values were normalized to GAPDH RNA levels for human cells and mouse kidney. Bcl-2, p21 and p53 were analysed with the TaqMan Gene Expression assay (Applied Biosystems). Thermal profiles used were previously published [51] (link), [52] (link). The primers for all the other mRNAs are listed in Table 4 of supplemental material.
3
HUVEC Total RNA Extraction and qRT-PCR
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Total RNA was isolated from HUVECs using Total RNA isolation kit (NorgenBiotek Corp, Thorold, Ontario, Canada) following the manufacturer’s instructions. First-strand complementary deoxyribonucleic acid (DNA) was prepared using 1–2 µg of total RNA, the Superscript III RT kit and random hexamer primers (Invitrogen, Carlsbad, CA, USA) in a total volume of 25 µl according to the manufacturer’s instructions. Reverse transcription reaction was carried for 90 min at 50 °C and an additional 10 min at 55 °C. Real-time PCR (qRT-PCR) was performed on an ABI Prism 7900 sequence detection system using Sybr Green reagents (Takara Bio Company, Clontech, Mountain View, CA, USA) and TaqMan® Gene Expression Master Mix (Life Technologies, Madrid, Spain).
4
Real-Time PCR Gene Expression Analysis
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Total RNA was extracted with the RNA purification kit (Norgen Biotek) and checked for concentration and purity with Nanodrop (Thermo Fisher). Sample with a 260/280 ratio of ~ 2.0 were selected and 1 μg of RNA was reverse-transcribed with Superscript III RT kit (Invitrogen) according to the manufacturer’s instructions. Real-time PCR (RT-PCR) was performed in a QuantStudio 6 Flex (Applied Biosystems) detection system using SybrGreen reagents (Takara Bio Company). For normalisation purposes, 18S was used as the reference gene [39 (link)]. The thermal profile used was as previously published [32 (link), 40 (link)]. The list of primers used can be found in Supplementary Table 1.
5
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated from HUVECs with a Total RNA Isolation Kit (Norgen Biotek Corp, Thorold, Ontario, Canada) in accordance with the manufacturer’s instructions. First-strand cDNA was prepared from 1-2 μg of total RNA, the Superscript III RT Kit and random hexamer primers (Invitrogen, Carlsbad, CA, USA) in a total volume of 25 μL, according to the manufacturer’s instructions. The reverse transcription reaction was carried out for 90 minutes at 50°C and for an additional 10 minutes at 55°C. Real-time PCR (qRT-PCR) was performed on an ABI Prism 7900 sequence detection system with SYBR Green reagents (Takara Bio Company, Clontech, Mountain View, CA, USA) and TaqMan® Gene Expression Master Mix (Life Technologies, Madrid, Spain).
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