Taqman array fast plate

RNA and cDNA were obtained as previously described from hippocampal samples (n = 4/experimental group) (Busquets et al. 2019c (link)). TaqMan® Array Fast plates (ThermoFisher Scientific, Inc.) were used to analyze a total of 48 genes. These were selected by their described relevance in the regulation of energetic metabolism and cognitive function.
18S, Actb, Gapdh, Hprt and Gusb were tested as housekeeping genes in all samples. Hprt showed the least variability and was thus selected to perform any analysis. Targets: Slc2a1, Slc2a2, Slc2a3, Slc2a4, Insr, Irs1, Irs2, Prkaa1, Akt1, Akt2, Creb1, Gsk3β, Pparγ, Pparγc1α, Ptpn1, Hk1, Hk2, Pfkp, Pkm, Pdha1, Pdha2, Ndufv1, Sdha, Sdhb, Uqcrc1, Uqcrb, Cycs, Cox4i1, Atp5b, Sod1, Gpx1, Cat, Bdnf, Ntrk2, Ppp1r9b, Syp, Dlg4, Nrxn1, Nrxn2, Nrxn3, Nlgn1, Nlgn2, Nlgn3. No data was reported of Slc2a2, Pparγ and Pdha2 since the TaqMan® probes produced either no signal or a C_T value over 35. Specific descriptions for each of the genes included in the study can be found in Additional file 1: S1.

Total RNA isolation was performed using a RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. RNA concentration and purity were determined using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific).
Reverse transcription was carried out with 250 ng of total RNA using the QuantiTect retrotranscription kit (Qiagen). Quantitative PCR (qPCR) reactions were performed using Taqman Array Fast plates and Taqman Gene expression master mix (Thermo Fisher Scientific) for CRX and BRN3B and S18 in an Applied Biosystems real-time PCR machine (7500 Fast System). All samples were normalized against a housekeeping gene (18S) and the gene expression was determined based on the ΔΔCT method relative to D35. Average values were obtained from at least four biological replicates. The primer sets and MGB probes (Thermo Fisher Scientific) labeled with FAM for amplification are listed in Table 3.

In order to identify the most prominent TRP channel candidates in the IVD, an initial set of eight IVD samples was used for the gene expression screening with TaqMan Array Fast Plates (Thermo Fisher, Switzerland). The screening sample set included four degenerated IVD samples (1x AF Pfirrmann Grade 4, 1x NP Pfirrmann Grade 4, 1x AF Pfirrmann Grade 5, 1x NP Pfirrmann Grade 5) and four non-degenerated IVD samples (2x AF, 2x NP).
The array was conducted according to the protocol provided by the manufacturer. Briefly, 180 µL of amplified cDNA (mixed with RNAse-free water) was combined with 180 µL of the TaqMan Fast Universal PCR Master Mix (2×) (Thermo Fisher, Switzerland) and added to a 96-well plate (10 µL per well), pre-coated with selected TaqMan primers, and gene expression was measured by the real-time qPCR (CFX96 Touch™ Detection System, Biorad). Each array constituted of 32 targets out of which 28 included human TRP channel targets: TRPA1, TRPC1–7, TRPM1–8, TRPML1–3, TRPP1–3, TRPV1–6, and PKD1, as well as three internal controls: 18S, YWHAZ and GUSB. Additionally, to identify the most stable reference gene, six additional internal controls (ACTB, GAPDH, RPL4, RPL13A, SDHA and TBP, see Supplementary Material) were tested on the IVD screening sample set and analyzed with the geNorm algorithm⁴⁷ .

To isolate pulmonary mouse MAIT cells, non-parenchymal lung mononuclear cells (LMNCs) from indicated B6-MAIT^CAST cohorts were subjected to magnetic cell sorting. Briefly, phycoerythrin (PE)-conjugated, 5-OP-RU-loaded mouse MR1 tetramers were used to stain MAIT cells [7 (link),42 (link)], which were then purified using Anti-PE MicroBeads UltraPure, LS Columns and a QuadroMACS Separator (Miltenyi Biotec). Isolated MAIT cells were always 90–99% pure after two separation rounds (S1 Fig). Cells were washed in PBS containing 10% BSA and 50 mM ethylenediaminetetraacetic acid before pellets were flash-frozen and stored at -80°C.
Total RNA was extracted using a PicoPure RNA Isolation Kit (Thermo Scientific) and converted to cDNA using SuperScript IV VILO Master Mix with ezDNase (Thermo Scientific). cDNA and TaqMan Fast Advanced Master Mix were added to each well of a custom-made, 96-well TaqMan Array Fast Plate (Thermo Scientific) containing lyophilized primer/probe sets listed in S1 Table. cDNA was amplified per manufacturer’s instructions, and cycle threshold (Ct) values were generated using a StepOne Plus Real-Time PCR System (Applied Biosystems). Normalized ΔCt values were determined by subtracting each Ct value by that of Gapdh, and the following formula was employed to calculate the relative mRNA content of MAIT cells for indicated genes: Fold Change = 2^-(ΔΔCt).

Total RNA was extracted using TRIzol from 1 to 2 × 10⁶ epSPCs cultured under the various conditions. RNA quality was verified using a 2100 Nano Bioanalyzer (Agilent Technologies, Waldbron, Germany). Total RNA was retrotranscribed using the SuperScript Vilo cDNA Synthesis kit. cDNA (corresponding to 100 ng total RNA) was amplified by quantitative (q)RT PCR, in triplicate, using Universal PCR master mix and TaqMan Array Fast Plate assembled by Thermo Fisher Scientific with primer and probe sets for the TERT, AKT1, AKT2, AKT3, UCP1, UCP2, PTEN, SOX2, and OCT4 genes and the 18S housekeeping gene on an Applied Biosystems PRIMS 7500 Fast Real-Time PCR System. mRNA expression levels were normalized against 18S, and the relative mRNA expression levels were calculated using the formula 2^−ΔCt.