Taqman ez rt pcr core reagents

The frozen liver samples (approximately 100 mg) were homogenized in ISOGEN solution (Nippon Gene Co., Ltd, Tokyo, Japan) and total RNA was extracted and purified using the RNeasy Mini Kit (QIAGEN K.K., Tokyo, Japan). mRNA expression levels were measured using a one‐step quantitative PCR with TaqMan EZ RT‐PCR Core Reagents (N808‐0236; Applied Biosystems™, Thermo Fisher Scientific K.K.). The primers for hamster CYP7A1 gene (Assay ID: HAMCYP7A1‐EX5) were designed and synthesized based on cDNA sequence using the Assay‐by‐design service (Applied Biosystems™, Thermo Fisher Scientific K.K.). The primers for hamster GAPDH gene (hamster G3PDH‐683F and hamster G3PDH‐748R) were purchased from Sigma‐Aldrich Japan and used as the reference gene. PCR reactions were performed using OPTICON2 (Bio‐Rad Laboratories, Inc., Tokyo, Japan) and gene expression was normalized to GAPDH expression.

Transfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The CXCR4 siRNA duplexes were purchased from Ambion (Austin, TX). Total mRNA was extracted using TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA by reverse transcription using Superscript III reverse transcriptase (Invitrogen), as described by the manufacturer. CXCR4 primers and probes were purchased from Applied Biosystems (Foster City, CA). EGFR-WT and EGFR-L858R were quantified by real-time RT-PCR (TaqMan) using the primers 5′-CCGCAGCATGTCAAGATCAC-3′ (forward) and 5′-TCCTTCTGCATGGTATTCTTTCTCT-3′ (reverse)49 (link) and the TaqMan probes 5′-VIC-TTGGCCAGCCCAA-MGB-3′ (EGFR-WT) and 5′-FAM-TTGGCCCGCCCAA-MGB-3′ (EGFR-L858R). Reactions were performed on an Applied Biosystems 7500 Real-Time PCR System using TaqMan EZ RT-PCR Core Reagents (Applied Biosystems).

Total RNA was prepared from cells by using an RNeasy mini kit (Qiagen). For quantitation of HCV-RNA, quantitative RT-PCR was performed by using TaqMan EZ RT-PCR Core Reagents and a ViiA7 system (Life Technologies) according to the manufacturer’s protocol. For quantitation of gene expression, the synthesis of the first-stranded cDNA was performed by using a PrimeScript RT Reagent Kit (Perfect Real Time) (Takara Bio) and quantitative RT-PCR was performed by using Platinum SYBR Green qRT-PCR SuperMix UDG (Life Technologies) according to the manufacturer’s protocol. ApoB, ApoE and MTTP were amplified using the primer pairs described previously [28 (link)]. For quantitation of miRNA, total RNA was prepared from cells by using an miRNeasy mini kit (Qiagen) and miR-122 was determined by using miR-122-specific RT primers and amplified by using specific primers provided in the Taqman MicroRNA Assays (Life Technologies) according to the manufacturer’s protocol. U6 small nuclear RNA (snRNA) was used as an internal control. Fluorescent signals were analyzed by using a ViiA7 system (Life Technologies).

Total RNA was extracted from cells by using an RNeasy minikit (Qiagen) and the first-strand cDNA synthesis and qRT-PCR were performed with TaqMan EZ RT-PCR core reagents and a ViiA7 system (Life Technologies), respectively, according to the manufacturer's protocol. The primers for TaqMan PCR targeted to the noncoding region of HCV RNA were synthesized as previously reported [54] (link). Taqman Gene expression assays were used as the primers and probes targeting to apolipoproteins (Life Technologies). Fluorescent signals were analyzed with the ViiA7 system.