Quantitative Real-Time PCR for Gene Expression

Total RNA was isolated using RNAqueous Isolation Kit (Ambion) according to the manufacturer's protocols. TaqMan® Gene Expression Assay primer and probe sets (Applied Biosystems) were used for real-time, quantitative PCR (qPCR) analysis of GAPDH (Hs99999905_m1), PKCζ (Hs00177051_m1), Par6 (Hs00180947_m1) and 18S (Hs99999901_s1). Forward and reverse primer and probe sequences for PKCι were previously described [2 (link)]. qPCR analysis was carried out using 10 ng of cDNA or 2 ng cDNA (18S) on an Applied Biosystems 7900 thermal cycler. Data was evaluated using the SDS 2.3 software package. Gene expression was normalized to GAPDH. All data is expressed as 2^{−(CT(target)−CT(endogenous reference))}.

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Butler A.M., Scotti Buzhardt M.L., Erdogan E., Li S., Inman K.S., Fields A.P, & Murray N.R. (2015). A small molecule inhibitor of atypical protein kinase C signaling inhibits pancreatic cancer cell transformed growth and invasion. Oncotarget, 6(17), 15297-15310.

Publication 2015

RNAqueous Isolation Kit TaqMan® Gene Expression Assay primer and probe sets 7900 thermal cycler

Assay Cdna Gapdh Gene expression Isolation Primer Quantitative real time pcr analysis

Corresponding Organization :

Other organizations : Mayo Clinic in Florida, Novartis (United States), Virginia Tech

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Gene expression levels of GAPDH, PKCζ, Par6, and 18S

control variables

Total RNA isolation using RNAqueous Isolation Kit (Ambion)
TaqMan® Gene Expression Assay primer and probe sets (Applied Biosystems) for qPCR analysis
CDNA input amount (10 ng for target genes, 2 ng for 18S)
Use of Applied Biosystems 7900 thermal cycler for qPCR
Data evaluation using SDS 2.3 software package
Gene expression normalization to GAPDH

controls

No positive or negative controls explicitly mentioned

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