Taqman array 96 well plate

SH-SY5Y cells were used for total RNA extraction, which was performed by using the RNAqueous ^®-4PCR kit (Ambion Inc., Austin, TX, USA) as previously reported [99 (link)]. Before cDNA synthesis, the integrity of RNA was evaluated by denaturing electrophoresis in TAE 1.2% agarose gel. cDNA was synthesized using 1 μg of total RNA for all samples using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) under the following conditions: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min for 40 cycles. RTq-PCR was performed using Master Mix TaqMan^®Gene Expression and the 7.500 RT-PCR instrument (Applied Biosystems), targeting genes in the Taqman Array 96-Well Plate (P/N: 4414292): brain-derived neurotrophic factor (BDNF, Hs02718934_s1), glial cell-derived neurotrophic factor (GDNF, Hs01931883_s1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs99999905_m1).

The quantitative real-time PCR (qPCR) reactions (20 μL) were performed with the TaqMan gene expression master mix (Life Technologies), and TaqMan array 96 well plates (Applied Biosystems, Foster City, CA) using the StepOnePlus^™ real-time PCR system (Applied Biosystems). The primers used are described in Table 1. The qPCR reactions were performed in triplicate. StepOnePlus^™ real-time PCR system (Applied Biosystems) was used for qPCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the samples to obtain cycle delta threshold over background (ΔC_T). The delta delta CT method (2^-ΔΔCT) was used to analyze the relative gene expression levels.

Expression of stemness genes was screening in ECFCs at P1 from 3 different cord blood samples with TaqMan Array 96-Well plates “Human Stem Cell Pluripotency” (Applied Biosystems, Fischer Scientific, Illkirch, France). The embryonic stem cell lines H9 at P45 and H1 at P54 (one sample of each) were used as calibrators. Two samples of mature differentiated cells were used as negative controls: One sample of HAECs from a 23-year old female donor and one sample of human skin fibroblasts from a 37-year old female donor, kindly provided by the human cell culture platform of the Myology Institute (Université Pierre et Marie Curie, Paris 6). Quantitative RT-PCRs were performed according to the manufacturer’s instructions using 7300 Real-Time PCR system (Applied Biosystems).

Quantitative Real-Time PCR experiments were performed in triplicate using optimised, validated human endogenous control assay TaqMan Array 96-Well Plates (Applied Biosystems by Life Technologies, Paisley, UK) that contain 32 reference genes (see Supplemental Table 1). TaqMan Gene Expression Assays were used (Applied Biosystems) and each consisted of a fluorogenic FAM dye–labelled MGB probe (final concentration 250 nM) and two amplification primers (forward and reverse; final concentration 900 nM) provided in a pre-formulated 20X mix. Each assay had an amplification efficiency of 100 ± 10%, calculated by the system software. RT-minus and no template controls (NTC) containing DNAse-free water instead of template mRNA were included in each run and no product was synthesised in the NTC and RT-minus reactions confirming the absence of contamination with exogenous DNA. The final reaction volume was 20 μl and consisted of 2 μl of cDNA, 8 μl of DNAse-free water and 10 μl of TaqMan universal PCR Master Mix. A StepOne Plus instrument (Applied Biosystems by Life Technologies, Paisley, UK) was used for the PCR with their proprietary software (StepOnePlus software version 2.3) automatically determining the Ct as being 0.5 standard deviations above baseline fluorescence. The cycle profile was 2 minutes at 50 °C, 10 minutes at 95 °C, 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C.