18s hs99999901 s1

Total RNA was extracted using the RNeasy® Mini Kit and accompanying QIAshredder™ (Qiagen). To avoid DNA contamination of samples, a 15 min on-column incubation was carried out with DNase I (Qiagen). Reverse transcription was performed using the SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific). For the quantitative analysis of gene expression we used the ABI Prism® 7000 Sequence Detection System or the StepOnePlus® Real-Time PCR System (Thermo Fisher Scientific). Target sequences were amplified from 50 ng of cDNA using the TaqMan® Primer and Probe assays for human eNOS (NOS3, Hs00167166_m1) and for the endogenous control 18 S (Hs99999901_s1) (Thermo Fisher Scientific), or the PrimeTime® qPCR assays for eNOS (NOS3, Hs.PT.58523162) and actin (ACTB, Hs.PT.39a.22214847) (IDT). For calculation of results, the 2^−ΔΔCt method was used.

RNA was isolated from tumor and normal tissue using TriZol™ Reagent (Life Technologies, Grand Island, NY, USA) and from plasma with TriZol™ LS Reagent (Life Technologies) supplemented with MS2 RNA using the Total RNA purification kit (Norgen, Thorold, ON, Canada) according to the manufacturer’s instructions. cDNA was obtained from 500 ng of RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Relative lincRNA-p21 expression was determined by real-time polymerase chain reaction (RTqPCR) using the StepOne™ Real-time PCR System Thermal Cycling Block (Applied Biosystems). Quantification of lincRNA-p21 expression levels was performed with Custom qPCR Probes (Integrated DNA Technologies, Coralville, IW, USA) using the primers and probes described by Hall et al. [17 (link)]. Taqman gene expression assays of B2M (beta-2-microglobulin) (Hs99999907_m1) and 18 s (Hs99999901_s1) (Applied Biosystems) were used as endogenous control for tissue and blood samples, respectively.

Each channel of the Brain-Chip was lysed using TRI Reagent® (Sigma- Aldrich, T9424) and RNA was isolated using the Direct-zol RNA Purification Kit (Zymo Research, R2060). Genomic DNA was removed using the TURBO DNA-freeTM Kit (ThermoFischer, AM1907) and reverse transcription to cDNA was performed with the SuperScript IV Synthesis System (ThermoFischer, 18091050). TaqMan Fast Advanced Master Mix (Applied Biosystems, 4444963) and TaqMan Gene Expression Assays were used for the Quantitative Real-Time PCR (qPCR). The qPCR was detected on a QuantStudio 3 PCR System (Fisher Scientific). The target genes were assessed using commercially available primers (GFAP: Hs00909233_m1, VIMENTIN: Hs00958111_m1, LCN2: Hs01008571_m1, CD68: Hs02836816_g1, EDA: Hs03025596_s1, APOA1: Hs00163641_m1, SNCAIP: Hs00917422_m1, LRRK2: Hs01115057_m1, MAOA: Hs00165140_m1). The results were quantified by the comparative Ct method. Ct values for samples were normalized to the expression of the housekeeping gene (18s: Hs99999901_s1; Applied Biosystems). All primers are listed in Supplementary Table 4.