Total RNA was isolated and extracted using TRIzol (Life Technologies, Carlsbad, CA) RNA was quantified for purity (2.0–2.1 A260/A280 ratio) and quantity using a NanoDrop Lite spectrophotometer (Thermo Scientific, Waltham, MA). RNA was then reverse transcribed to 0.5–2ug of complementary DNA (cDNA) using high capacity cDNA reverse transcription kit (Applied biosystems, #4374966, Waltham, MA). Real-time PCR results were obtained using the CFX Opus 96 system an iQ SYBR Green Supermix (Biorad, Hercules, CA) with 4ng of cDNA template and the final primer concentration was 0.5 uM. Expression levels were quantitated and normalized to GAPDH. The following primers were used for amplification of target cDNA: TNF-α, 5’ TGA GGT ACA GGC CCT CTG AT 3’ and 5’ CTC GAA CCC CGA GTG ACA AG 3’; IL-8, 5’ GGC CGT GGC TCT CTT GGC AG 3’ and 5’ TGT GTT GGC GCA GTG TGG TCC 3’; GAPDH, 5’ TCA AGG CTG AGA ACG GGA AG 3’ and 5’ CGC CCC ACT TGA TTT TGG AG 3’.
Cfx opus 96 system
Manufactured by Bio-Rad
Sourced in Germany, Japan
The CFX Opus 96 system is a real-time PCR detection platform designed for gene expression analysis, genotyping, and other quantitative PCR applications. The system features a 96-well block format and utilizes LED-based optical detection technology to accurately measure fluorescent signals during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Blue s green master mix, by Biozym (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Applied biosystems fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Nucleospin rna plus xs kit, by Macherey-Nagel (1 mentions)
Thunderbird next sybr qpcr mix, by Toyobo (1 mentions)
Trizol extraction protocol, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
5 protocols using cfx opus 96 system
1
Quantitative mRNA Expression Analysis
2
Quantifying Hypoxia-Responsive Genes by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using the NucleoSpin RNA kit (740955.250, Macherey-Nagel, Düren Germany) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 500 ng total RNA using M-MLV reverse transcriptase (M1705, Promega, Walldorf, Germany) and oligo dT primer. Quantitative Real-Time PCR (qRT-PCR) was performed with a Biozym Blue S’Green master mix (331416XL, Biozym Scientific, Hessisch Oldendorf, Germany) on a BioRad CFX Opus 96 System. Relative expression levels were calculated with the ΔΔct method using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reference gene. The following primer pairs were used: HIF1A 5′-GGATGCTGGTGATTTGGATA-3′ (forward) and 5′-TCATGGTCACATGGATGAGTA-3′ (reverse); HIF2A 5′-CGGAGGTGTTCTATGAGCTGG-3′ (forward) and 5′-AGCTTGTGTGTTCGCAGGAA-3′ (reverse); CA9 5′-CACGTGGTTCACCTCAGCAC-3′ (forward) and 5′-CAGCGATTTCTTCCAAGCG-3′ (reverse); HPRT 5′-CCTGGCGTCGTGATTAGTGA-3′ (forward) and 5′-CGAGCAAGACGTTCAGTCCT-3′ (reverse).
3
RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellets were immediately processed using the NucleoSpin RNA Plus XS kit (Macherey-Nagel) or the RNAeasy kit (Quiagen) as per manufacturer’s instructions. 1μg eluted RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo-Fisher). Quantitative polymerase chain reactions were performed using Applied Biosystems Fast SYBR Green Master Mix (Applied Biosystems) on a CFX Opus 96 system (BioRad). HPRT1 was used as a house-keeping gene and the 2−ΔΔCT method to calculate differences in RNA levels.
4
Quantitative Real-Time PCR Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from VSMCs or vascular tissues following the TRIzol extraction protocol (Invitrogen). Equal amounts of RNA were reverse transcribed into cDNA using the Prime Script RT Reagent Kit (TAKARA, Dalian, China). qRT-PCR amplification was performed with the THUNDERBIRD® Next SYBR qPCR Mix (TOYOBO, Osaka, Japan) on a CFX Opus 96 system (Bio-Rad, CA, USA). The mRNA levels were normalized to those of GAPDH. Data were analyzed using CFX Maestro qPCR analysis software version (V2.3). The primer sequences are listed in Supplementary Table S1 .
5
Quantifying Gene Expression in Cells
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!