Taqman low density array

In brief, striatal mRNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) and then reverse transcribed and amplified with the TaqMan One-Step RT-PCR master mix kit (Life Technologies, CA) according to the manufacturer’s instructions. The relative mRNA levels of the target genes were determined by quantitative real-time PCR using a microfluidic card TaqMan^® Low Density Array (TLDA) on an ABI Prism 7500 (Life Technologies, CA). The expression levels were normalized using DataAssist v2.0 Software (Life Technologies, CA). The endogenous controls used for analysis were β-actin, GAPDH, hypoxanthine-guanine phosphoribosyltransferase-1 (Hprt1) and mouse 18S mRNAs.

An a priori list of splicing factor candidate genes were chosen on the basis that they were associated with human aging in populations and in primary human cell lines that had undergone in vitro senescence in our previous work (Harries et al., 2011; Holly et al., 2013). The list of genes included the positive regulatory splicing factors Srsf1, Srsf2, Srsf3, Srsf6, Srsf18 and Tra2β, the negative regulatory splicing inhibitors Hnrnpa0, Hnrnpa1, Hnrnpa2b1, Hnrnpd, Hnrnph3, Hnrnpk, Hnrnpm, Hnrnpul2 and the Sf3b1 subunit of the U2 spliceosome snRNP, which we have previously shown to be associated with age‐related altered DNA methylation (Holly et al., 2014). Assays were obtained in custom TaqMan low‐density array (TLDA) format from Life Technologies (Foster City, CA, USA). Assay Identifiers are given in Data S1.

TaqMan® Low Density Array (TLDA) was used (Life Technologies, Carlsbad, CA) to examine the expression of 46 human DNA repair genes in 10 GSCs before and 3 h following 4Gy. The list of target genes is detailed in Additional file 2: Table S1. Two microgram of total RNA were reverse transcribed using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s instructions (Life Technologies). Real-time PCR experiments were then carried out with the ABI PRISM 7900HT Sequence Detection System. Each experiment was conducted in triplicate. Relative quantification (RQ) of target gene expression was determined by the 2^−ΔΔCt method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH, most stable reference gene) as an endogenous control. Data were analyzed using the StatMiner 3.0 software (Integromics, Madrid, Spain).

Total RNA was isolated from xenografted tumors at a size of approximately 100–300 mm³ using an RNeasy kit (Qiagen, Valencia, CA). Reverse transcription was performed for 59 genes related to angiogenesis using Multiscribe™ Reverse Transcriptase (Invitrogen), and the quantitative real-time polymerase chain reaction (qPCR) analysis was performed using a TaqMan® Low-Density Array (Invitrogen) (Additional file 1).

31 biopsies from from patients (n = 29) with initial UA with knee synovitis were included in this part of the study. A repeat biopsy at 3 months was collected in 2 patients who had received placebo treatment in the context of a randomized trial. 30 of these samples were also hybridized on the low-density microarrays. All patients were untreated at the time of the biopsy. A clinical diagnosis was obtained in all of them after a median follow-up of 9 months: RA (n = 15), OA (n = 11), and SA (n = 5).
Total RNA (500 ng) was reverse-transcribed into cDNA, using the high-capacity reverse transcription kit (Life Technologies, Merelbeke, Belgium). A customized Taqman low-density array (Life Technologies) was designed in order to assess the expression of 95 signature and one house-keeping (18s) genes, using pre-designed Life Technologies Taqman assays. 16 ml cDNA was mixed with 84 ml H2O and 100 ml 2x Taqman universal master mix (Life Technologies). Each sample was loaded into two ports of the card, and run on an ABI 7900HT system for 2 min at 50°C, 10 min at 95°C, followed by 40 cycles for 15s at 97°C and 1 min at 60°C. One of the assays (MYEOV2) failed to generate any signal, and was excluded from the analyses. Raw qPCR data are displayed in S2 Table.