Total RNAs of human tissues and cultured cells were isolated using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction. The quality and concentration of extracted RNAs were determined by collecting the absorbance at 260 nm with Nanodrop 2000. The RNAs were immediately transcribed into cDNAs using the PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Shiga, Japan). All real-time PCRs were performed with the SYBR Premix Ex Taq Kit (TaKaRa, Japan) in an ABI PRISM 7500 Real-Time System. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as the internal control. Each experiment was repeated three times with each one performed in triplicate.
Abi prism 7500 real time system
Manufactured by Takara Bio
Sourced in Japan
The ABI PRISM 7500 Real-Time System is a laboratory instrument designed for real-time PCR analysis. It is capable of detecting and quantifying nucleic acid sequences in real-time during the amplification process.
Automatically generated - may contain errors
2 protocols using abi prism 7500 real time system
1
RNA Extraction and qRT-PCR Analysis
2
Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, US), and followed by cDNA synthesis with Taq-Man Reverse Transcription Kit (Takara, Dalian, China) according to manufacturer’s instructions. mRNA expression was detected by SYBR Premix Ex Taq kit (Takara) performed on an ABI Prism 7500 Real-Time System, and relative mRNA amount was normalized against β-actin and calculated by 2-ΔΔCt. Sequences of PCR primers were referred from online PrimerBank (https://pga.mgh.harvard.edu/primerbank/ ) and listed in Additional file 1 : Table S1.
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