Total RNA was isolated from LX-2 cells using Trizol Reagent (Invitrogen) and quantified. RNA was synthesized from 1 μg of total RNA using cDNA Reverse Transcription kit (Invitrogen). Real-time PCR was performed using the Mastercycler ep realplex 4 real-time PCR system (Eppendorf) with an SYBR Green qPCR Master Mix (Fermentas) according to the manufacturer's protocol. The sequences of primers for human α-SMA (NC_000010.11) were 5′-CCACCGCAAATGCTTCTAAGT-3′ (forward) and 5′-GGCAGGAATGATTTGGAAAGG-3′ (reverse). The primers for human GAPDH (NM_002046.3) were 5′-TGCACCACCAACTGCTT AGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse). The amplification conditions were as follows: 951C for 10 min, and 40 cycles of 951C for 15 s, 641C for 30 s and 721C 20 s. The amount of α-SMA transcripts of individual samples was normalized to GAPDH.
Mastercycler ep realplex 4 real time pcr system
Manufactured by Eppendorf
The Mastercycler ep realplex 4 is a real-time PCR system. It is capable of running quantitative PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Avian myeloblastosis virus reverse transcriptase, by Promega (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
2 protocols using mastercycler ep realplex 4 real time pcr system
1
Quantification of α-SMA mRNA Expression
2
Quantification of Human B7-H1 Expression
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RNA was treated with RNase-free DNase-I (Takara Bio) and reverse-transcribed using avian myeloblastosis virus reverse transcriptase (Promega). Real-time PCR was performed using the Mastercycler ep realplex 4 real-time PCR system (Eppendorf) with an SYBR Green qPCR Master Mix (Fermentas), according to the manufacturer's protocol. The sequences of primers for human B7-H1 (NM_ 014143.3) were 5′-TGCGTTCAGCAAATGCCAGT-3′ (forward) and 5′-ATTGCAGGATGCAGGGGTGTA-3′ (reverse). The primers for human GAPDH (NM_002046.3) were 5′-TGCACC ACCAACTGCTTAGC-3′ (forward) and 5′-GGCATGGACT GTGGTCATGAG-3′ (reverse). The amplification conditions were as follows: 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, 63 °C for 30 s, and 72 °C 20 s. The amount of B7-H1 transcripts of individual samples was normalized to GAPDH.
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