Taqman low density array cards

Gene expression analysis was carried out using Taqman Low-Density Array cards (Life Technologies) on an ABI7900HT instrument (Applied Biosystems, Life Technologies). Relative gene expression was determined using the ΔΔC_T method, with expression levels normalised to the geometric mean of three housekeeping genes (GAPDH, PPIA and UBC). TaqMan probes used are listed in Supplementary Table 1. All samples were amplified in duplicate and data are presented as arbitrary units.

Isolation and miRNA profiling were described previously [62 (link)]. In short, trypsinized cells were lysed in Lysis/Binding buffer and miRNA was isolated according to the protocol of miRVANA Isolation Kit (Applied Biosystems, Vilnius, Lithuania). TaqMan® MicroRNA Reverse Transcription Kit and Custom RT Primer Pool (Applied Biosystems, Foster City, CA, USA) were used in reverse transcription reaction. Expression profiling of miRNAs was carried out with TaqMan Low-Density Array (TLDA) cards (Applied Biosystems, Foster City, CA, USA) using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). miRNA expression data were determined from duplicate results, normalized using the 2−∆Ct method with the arithmetic average of Ct values for reference genes let-7b-5p (002619) and U6 (001973) selected using RefFinder software. The presented miRNAs were chosen based on screening analysis, bioinformatics analysis as well as literature search (miR-26a-5p assay ID 000405, miR-26b-5p assay ID 000407, let-7d-5p assay ID 002283, let-7e-5p assay ID 002283, miR-365a-3p assay ID 001020, miR-146a-5p assay ID 000468, miR-378a-3p assay ID 002243, mir-193b-3p assay ID 002367; Applied Biosystems, Foster City, CA, USA).

Total RNA was isolated from 1 × 10⁶ tumor-tissue derived cells using the RNA Easy Mini Kit (Qiagen) according to the manufacturer’s instructions. The concentration and purity of the samples were determined by spectrophotometry with a NanoDrop© 2000c (Thermo Scientific), and the RNA integrity was assessed using a 2100 Bioanalyzer (Agilent). Complementary DNA was synthesized from 100 ng of total RNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). The gene expression of immune response-associated genes was determined using TaqMan low-density array (TLDA) cards according to the manufacturer’s instructions (Applied Biosystems). The TLDA cards (TaqMan® Array Human Immune Panel) were run on a Viia7 instrument (Applied Biosystems) using TaqMan® Universal Master Mix II, no UNG (Applied Biosystems). Ct values were analyzed using GenEx software (MultiD Analyses). Relative gene expression levels were calculated using the ΔΔCt method and were normalized to the expression levels of reference genes GUSB and TFRC, selected by GeNorm from 6 reference genes assessed in total.

RNA was purified from fresh frozen tumor tissue and synthesis of cDNA was performed from 1 μg of total RNA using the High-Capacity RNA-to-cDNA Kit (cat. 4387406, Applied Biosystems; Foster City, CA). qPCR was performed using Taqman Low-Density Array (TLDA) Cards (cat. 4346800, Applied Biosystems) (Supplementary Table 2). qPCR Ct values were determined with predefined thresholds that were equal per gene for all patients. Relative gene expression was determined by the ΔΔCt method [28 ] using GUSB Ct-values for normalization. GUSB was selected as the most stable housekeeping gene (see Supplementary Table 7) out of four candidate genes (GAPDH, GUSB, RPLP0, and RPL4).

We monitored the mRNA expression levels of 24 mRNA transcripts, using TaqMan Low-Density array (TLDA) cards from Applied Biosystems (UK). Eighteen of the transcripts were analyzed in both studies (Additional file 1; overview of mRNA transcripts). mRNA transcripts were selected based on a thorough literature search investigating the effect of acute exercise on gene expression levels in PBMCs [36 (link)] as well as skeletal muscle [37 (link)]. Moreover, the selection was based on studies where effects of dairy products on markers of inflammation were investigated [38 (link)]. TLDA cards were used on a 7900 HT Applied Biosystems RT-qPCR machine (Applied Biosystems, UK). The Ct values were analyzed using SDS 2.4 (Applied Biosystems, UK), and further transferred to ExpressionSuite Sofware v1.0.3 (Applied Biosystems, UK). We normalized the Ct values to TATA box-binding protein (TBP) mRNA transcripts. Fold changes in mRNA transcripts from baseline to after the exercise session were calculated by dividing 2^{−ΔCtpost exercise} with 2^{−ΔCtbaseline}, using the 2^−ΔΔCt-method [39 (link)].