Taqman microfluidic cards

One million NPCs were pelleted for RNA extraction and qRTPCR analysis. For neuronal differentiation, 4x 10⁵ NPCs were seeded into one well of a 6 well plate for 4 weeks, and were then collected for RNA extraction. RNA was extracted using the RNeasy Mini kit (Qiagen) and reverse transcribed using the High Capacity RNA to cDNA kit (Thermo Fisher Scientific). For general differentiation marker analysis, qPCR was carried out using custom designed Taqman microfluidic cards (Thermo Fisher Scientific) containing 22 pluripotent, multipotent, neuronal and glial markers, as well as endogenous controls 18s ribosomal RNA and GAPDH[30 (link)]. Standard Taqman qPCR was used for the analysis of cellular stress-associated genes CDKN1a, SCD, CCNG2 and DDIT3, using ActB as endogenous control.

Cytokine mRNA measurement following stimulation of cells and skin was performed by qRT-PCR. Total RNA was prepared from cells lysate using TRIzol (Invitrogen, Carlsbad, CA) as per manufacturer instructions. Contaminating genomic DNA was removed with TurboDNase (Ambion, Austin, TX). Subsequently RNA (1 μg/mL) was reverse transcribed into cDNA using high capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster City, CA) as per manufacturer recommendation. The qRT-PCR reactions were conducted in 1X Universal master mix (Applied Biosystem, Foster city, CA) with 1/20 volume cDNA/reaction for individual gene expression assays, in a Viia7 Real-time PCR system (Applied Biosystem, Foster city, CA). Custom-made human specific TaqMan® Micro Fluidic Cards containing panels of 96 gene expression assays were loaded with a sample volume of cDNA solution equivalent to 250 ng of the original RNA, mixed with 2x Universal Master Mix (ThermoFisher, Carlsbad, CA) The Ct values obtained for each gene were directly normalized to housekeeping values (GAPDH or 18S) in order to obtain ΔCt. Change in the expression levels of mRNA in stimulated cultures were indicated as fold increase over media treated cells using 2^−ΔΔCt.

TaqMan microfluidic cards (Applied Biosystems) were processed as described by the manufacturer’s instructions. In brief, 100 ng of cDNA was mixed with TaqMan Universal PCR Master Mix (Applied Biosystems) before being injected into the microfluidic cards and dispersed into the wells by centrifugation. Microfluidic cards were sealed, and qPCR assays were run on the 7900HT Fast Real-Time System (Life Technologies) using ABI PRISM 7900 Sequence Detection System software (v2.4; Applied Biosystems, Life Technologies). The cycle thresholds were analysed using DataAssist Software (v2.0). Hierarchical clustering of gene expression and heatmap representation was performed with dChip software (Harvard).