Total RNA was isolated from aorta using TRI reagent (Molecular Research Center, Inc) according to the manufacturer’s instructions. 2 μg of total RNA were used to synthesize first stranded cDNA with a High-Capacity cDNA Reverse Transcription Kits. PCR was performed according to the manufacturer’s protocol using ABI PRISM® 7000 Sequence Detection System 26 (Applied Biosystems, CA) and QuantiFast SYBR Green PCR Kit (Qiagen, CA). Amplification conditions were performed with a 5 min preincubation at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. PCR products were subjected to melting curve analysis, using the ABI PRISM® 7000 Sequence Detection System, to exclude amplification of unspecific products. All real-time PCR primers were purchased from predesigned primers of QuantiTect primer assays (Qiagen). Results were normalized by 18S or HPRT expression levels.
Abi prism 7000 sequence detection system 26
Manufactured by Thermo Fisher Scientific
The ABI PRISM® 7000 Sequence Detection System is a real-time PCR instrument designed for gene expression analysis and other applications. It utilizes fluorescence detection technology to monitor DNA amplification in real-time. The system provides accurate and sensitive quantification of target DNA sequences.
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2 protocols using abi prism 7000 sequence detection system 26
1
Quantitative Analysis of Gene Expression
2
Quantitative Real-Time PCR Protocol
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Total RNA was isolated by using Tri Reagent (Molecular Research Center, Inc.) and phenol/chloroform. Reverse transcription was carried out using a high-capacity cDNA reverse transcription kit (Applied Biosystems) with 2 µg of total RNA. The PCR was performed according to the manufacturer’s protocol using ABI PRISM 7000 Sequence Detection System 26 (Applied Biosystems, CA) and QuantiFast SYBR Green PCR Kit (Qiagen, Valenica, Foster City, CA). Primer sequences for qPCR are listed in Supplementary Table 1 . Samples were all run in triplicates to reduce variability. The expression of genes was normalized and expressed as fold changes relative to HPRT or18S.
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