Taqman array card

The genotyping of the 10 bioinformatically determined splice SNPs was performed with the DNA extracted from the blood samples. For the genotyping, we used custom TaqMan^® Array Cards (Applied Biosystems). Data analysis was performed in an automated manner using the TaqMan Genotyper Software (version 1.6, Applied Biosystems). The genotype assignments were manually validated. In case of failed genotyping, the SNP was not considered for further analyses.

Specific reverse transcription of 150 ng of total RNA was performed using Megaplex™ RT Primers Human Pool A v2.1 (Step 1) or Custom RT Primers Human Pool (Step 2 and 3) (Thermo Fisher Scientific, Les Ulis, France), on a Veriti Thermal Cycler (Applied Biosystems™, Thermo Fisher Scientific). No complementary (c)DNA preamplification was required. miRNA expression was assessed by qPCR, using TaqMan^® Array Cards (Applied Biosystems™, Thermo Fisher Scientific), where the primers and probe of each miRNA were spotted and dried in duplicate wells of a microfluidic card. We used TaqMan^® Array Human MicroRNA A+B Card Sets v3.0 for the discovery cohort (a two-card set enabling quantitation of 754 unselected human miRNAs) and Custom TaqMan^® Array MicroRNA Cards for the selection and validation cohorts. Data analysis was performed by using Expression Suite software version 1.0.3. Assays with a cycle threshold (CT) values >35 were considered to be not expressed. RNU44 and RNU48 showed consistent expression in all the samples [mean (RNU44+RNU48) coefficient of variation was 3.73% in Array Card A and 3.79% in Array Card B] and were used as housekeeping genes for data normalization across all sub-studies.

Total liver RNA was isolated using PureLink RNA Mini kit (ThermoFisher, catalogue number 12183018 A), and quantified using the Qubit RNA BR Assay Kit (ThermoFisher Scientific, catalogue number Q10210) and a Qubit 3.0 Fluorimeter (ThermoFisher Scientific, catalogue number Q33216). The extracted RNA (1 μg) was reverse transcribed to cDNA using Superscript VILO cDNA synthesis kit (Invitrogen, catalogue number 11756050).
The expression of 87 genes was measured, in 5 livers per group, by qPCR using custom TaqMan array cards (format 384-well microfluidic card, Applied Biosystems, Foster City, CA), which were pre-spotted with custom designed dried-down TaqMan™ probes including 3 controls, 18S, Hprt and Rpl37a, as listed (Supporting information 4). Real time qPCR used the QuantStudioTM 12 K Flex Real-Time PCR System (Applied Biosystems) and Expression Suite v1.0 (Applied Biosystems), utilizing the comparative Cτ (ΔΔCτ) method for data analyses. Gene expressions were normalized to the 3 endogenous controls.

We performed a molecular screening array based on TaqMan Array Cards (Life Technologies, USA), allowing analysis of selected genes related to both angiogenesis and inflammation. We also used TaqMan gene assay for analysis of EZH2 (Hs01016789_m1) and TERT (Hs00972650_m1). In addition, we performed real-time PCR using cDNA as the template in a 20 μl reaction mixture containing SYBR Select Master Mix (Life Technologies, USA) and a specific primer pair for the following genes: OCT4, SOX2, NANOG, PPARG, FABP4, LPL, OC, OPN, ALP, SOX9, ACAN, COL2A1, and GAPDH (Table 1). Total RNA was extracted with the RNeasy Mini Kit and treated with DNAse (QIAGEN, Hilden, Germany). Subsequently, 100 ng of RNA was transcribed with the high-capacity RNA-to-cDNAkit protocol (Life Technologies, USA) in order to produce single-stranded cDNA. Samples were analyzed using the ABI Prism 7900HT Real-Time PCR System (Life Technologies, USA). The GAPDH gene was used as housekeeping gene. Furthermore, hierarchical cluster analysis of gene expression was used to group treatments with a similar expression pattern. Gene expression data of 34 genes assayed with TaqMan Array Cards were grouped using a hierarchical clustering algorithm in the Gene Cluster 3.0 program. A heat map was generated using Java TreeView program.

Pathogen screening was performed on Fast-track Diagnostics (viral and bacterial gastroenteritis and stool parasite kits) (manufactured by Siemens), Allplex (GI-Parasite and GI-Helminth(I) assays manufactured by Seegene) as well as TaqMan Array Cards (TAC, manufactured by Life Technologies) (included pathogens indicated in S1 Table). Tests were done according to the manufacturer’s instructions. Monoplex PCR was used to determine final outcome where there were discrepant results between testing platforms for a specific target. All tests included internal controls for sample validation. Due to limited availability of TAC cards, cases with unknown HIV status were excluded from testing. Controls with unknown HIV status were included in the overall case-control analysis (since there were limited control specimens available) but were not included in the HIV specific case-control analysis.

MicroRNA expression profiling was performed using Taqman™ MicroRNA Array Human Panel A + Panel B v2.1 (Life Technologies, Carlsbad, CA, USA) in order to evaluate the expression of 768 microRNAs. Megaplex™ Reverse Transcriptase reaction, was performed according to the manufacturer’s protocols (Life Technologies) using 500 ng of total extracted RNA. ViiA7 PCR instrument platform was used to analyze Taqman array cards and Expression Suite 2.1 software (Life Technologies, Carlsbad, CA, USA) was used to evaluate amplification plot efficiencies and to analyze data. Analysis was performed by using 2^−∆Ct method following normalization with small nuclear RNAs, RNU44 and RNU48.
Hierarchical clustering analysis plot was computed in order to obtain a global view of microRNA expression levels and to identify clustered group of microRNAs. Differentially expressed microRNAs were identified by performing a Volcano-plot analysis by applying a cut-off fold change of >2.5 (upregulated) or <0.4 (downregulated) and a statistical cutoff of FDR corrected p-value of p < 0.05 using Student’s t-test on normalized ∆Ct values. Hierarchical clustering analysis plot and volcano plot were elaborated using Spotfire 5.0 (Tibco, Somerville, MA, USA) and GraphPad 5.0 (GraphPad Prism, La Jolla, CA, USA), respectively.

Procedures for sample extraction and testing have been previously detailed [13 (link), 14 (link)] and the protocols are available at dx.doi.org/10.17504/protocols.io.5qpvo3k8xv4o/v1. We used custom-designed TaqMan Array Cards (ThermoFisher, Carlsbad, CA, USA) that compartmentalized probe-based quantitative PCR assays for 34 enteropathogens (S1 Table). A cycle threshold of 35 was used as the limit of detection and any detections above that were considered positive. Samples were stored at –80°C before extraction. Bacteriophage MS2 and phocine herpesvirus were used as external controls to monitor efficiency of nucleic acid extraction and amplification. We included one extraction blank per batch and one no-template amplification control per ten cards to exclude laboratory contamination.