Rt2 ht first strand kit

The levels of FXN transcript were measured with a SYBR green-based qRT-PCR assay. For whole blood analysis, total RNA was isolated from blood collected with PAXgene Blood RNA tubes and processed using PAXgene Blood RNA Kit IVD (QIAGEN). For RG2833 in vitro treated PBMC analysis, PBMCs were isolated from predose patient blood with Ficoll gradient and treated with RG2833 in culture for 24 hours. Cells were then pelleted, and RNA was isolated from the cell pellets using the RNeasy Mini Kit (QIAGEN). cDNA was then synthesized using the RT2 HT First Strand Kit (SABiosciences, Valencia, CA). Twenty-five nanograms of cDNA was combined with RT2 FAST SYBR Green/ROX qPCR master mix, RT2 qPCR primer assay for FXN (PPH05744B, SABiosciences), and RT2 qPCR primer assay for TBP (PPH01091G, SABiosciences) in a 25μl reaction volume. Assays were performed in duplicate using an Mx3005 or Mx3000 instrument and analyzed using MxPro Software (Agilent Technologies, Santa Clara, CA). The amount of FXN mRNA in each sample was determined relative to the predose sample and normalized to the levels of TBP mRNA.

For analysis of synovial tissue gene expression, total RNA was isolated from biopsies of 19 patients with RA using RNA STAT-60 (Invitrogen) according to the manufacturer’s instructions, and cleaned using RNeasy columns (Qiagen) and DNAse treatment. RNeasy Micro Kit (Qiagen) was used for RNA extraction from FLS. Quantity and purity of RNA was assessed using a Nanodrop spectrophotometer (Nanodrop Technologies). About 150–500 ng of RNA was reverse transcribed using a First-Strand cDNA synthesis kit (Thermo Scientific) and quantitative (q)PCR reaction was performed using Fast SybrGreen PCR Master Mix (Applied Biosystems). Sequences of the primers used are listed in online supplementary table 1. For qPCR array analysis, 1000 ng of RNA was reverse transcribed using an RT² HT First Strand Kit (Qiagen), cDNA was mixed with SybrGreen qPCR Mastermix (Qiagen) and expression of 84 genes involved in FLS activation was analysed using RT² Profiler customised qPCR arrays. qPCR reactions were performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) and relative mRNA expression was calculated using StepOne Software V.2.1 (Applied Biosystems) as the ratio between the gene of interest and the expression of housekeeping gene(s) using the ΔΔCT method.

The extracted RNA was converted to cDNA using the RT² HT first strand kit from Qiagen (cat # 330401). The cDNA was stored at −20°C until used for real-time PCR profiling using the mitochondrial energy metabolism PCR array from Qiagen (# PAHS008Z).