Dilutions of the cDNA (0.5 μL of cDNA per 25 μL reaction) were used as templates in a qPCR reaction containing QuantiTect 2× SYBR Green PCR Master Mix, gene-specific primers, and RNase-free water, using the standard protocol.
For qPCR analysis, cDNAs were added to reactions containing Taqman PCR master mix, gene-specific primer-probe sets for Col1α1, Tgfβ, Vegfα, Tnc, IL-6, IL-1β, Cxcl-2, Anf, Serca2α, Ryr2, Pgc1α and Gapdh (Applied Biosystems), and amplified using an ABI-7500-PCR ABI-7500-PCR ABI-7500-PCR
For single-gene expression measures, quantitative real-time PCR (qPCR) was carried out using TaqMan universal PCR master mix and gene-specific TaqMan gene expression probes (ABI).
Real-time PCR assays were performed in quadruplicate using TaqMan Gene Expression Master Mix (ABI), gene-specific TaqMan TAMRA probes (ABI) and an ABI 7000 sequence detection system.
PCR was performed in triplicate in TaqMan Universal PCR master mix containing gene specific probes or endogenous control (Applied Biosystems).
Quantitative real-time PCR was carried out with TaqMan PCR Master Mix, TaqMan gene expression assays, and an Applied Biosystems 7900HT thermal cycler.
First strand cDNA samples from 18 different human tissues were purchased from OriGene, Inc. Universal human cDNA was purchased from BioChain Institute, Inc. Taq Man Universal Master Mix, TaqMan PreAmp Master Mix and gene expression assays were from Applied Biosystems.
For all experiments unless otherwise stated, 20-μL reactions containing 5 μL sample were performed using universal master mix or gene expression master mix (Table S2) and the 7900HT fast real-time PCR system (all Life Technologies).
PCR was performed with the EmeraldAmp MAX PCR Master Mix and gene-specific primers.
About 2 µL of cDNA were amplified using the TaqMan® Gene Expression Master Mix and TaqMan® Gene Expression Assay for frataxin (Applied biosystems, catalog n°Hs00175940_m1) in a StepOne real-time PCR.
