Five ml of cleared secretome was 1:1 diluted in PBS before ultracentrifugation at 100,000× g for 9 h at 4 °C. Resulting pellets were processed with miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany) after addition of 30 pg of exogenous Arabidopsis thaliana ath-miR-159a synthetic miRNA spike. This was used to evaluate RNA recovery and cDNA synthesis performed as previously reported [14 (link)]. The OpenArray system (Life Technologies, Foster City, CA, USA) was used to determine miRNA expression in 384-well OpenArray plates according to the manufacturer’s protocol. Each single miRNA was considered as present when amplification resulted in all three samples. Eventually, ath-miR-159 spike-in CRT was used to equalize technical differences, and the global mean method [15 ] allowed normalization between samples.
Openarray system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The OpenArray system is a versatile lab equipment designed for high-throughput gene expression analysis. It enables the processing of multiple samples simultaneously, allowing for efficient and accurate data collection.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy and rneasy cleanup kits, by Qiagen (3 mentions)
Rneasy cleanup kit, by Qiagen (2 mentions)
Openarray, by Thermo Fisher Scientific (1 mentions)
Type 70.1 ti fixed angle titanium rotor, by Beckman Coulter (1 mentions)
Optima l 90k ultracentrifuge, by Beckman Coulter (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Taqman snp technology, by Thermo Fisher Scientific (1 mentions)
Mirneasy, by Qiagen (1 mentions)
Openarray plates, by Thermo Fisher Scientific (1 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Openarray accufill system, by Thermo Fisher Scientific (1 mentions)
Dataassist software, by Thermo Fisher Scientific (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
10 protocols using openarray system
1
Extracellular RNA Quantification Protocol
2
Profiling Extracellular Vesicle miRNAs
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Secretomes were 1:1 diluted in PBS for a total volume of 10 mL and ultra centrifugated (100,000× g, 9 h at 4 °C) in an Optima L-90K Ultracentrifuge (Beckman Coulter) equipped with a Type 70.1 Ti Fixed-Angle Titanium Rotor (Beckman Coulter). To evaluate the efficiency of RNA recovery and cDNA synthesis, an exogenous Arabidopsis thaliana ath-miR-159a (30 pg) synthetic miRNA spike was added to EV pellets before total RNA extraction with miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany). The OpenArray system (Life Technologies, Foster City, CA, USA) equipped with the 384-well OpenArray plates was used to detect the presence of 754 miRNAs, according to the manufacturer’s instructions. Each single miRNA was considered as present and considered for further analyses only when amplification appeared in all three samples. The equalization of technical differences was performed scoring ath-miR-159 spike-in CRT. Eventually, the global mean method allowed normalization between samples.
3
EV Small RNA Profiling by OpenArray
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Total RNA was isolated from similar EV numbers (10 ± 3 E9) using the miRNeasy Kit and RNeasy Cleanup Kit (Qiagen, Hilden, Germany), following the manufacturer’s instruction. To monitor the RNA recovery procedure, before RNA extraction, samples were spiked-in with exogenous ath-miR-159a (30 pg), an Arabidopsis thaliana synthetic miRNA whose specific primers are provided in the RT and PreAmp primer pools (Life Technologies, Foster City, CA, USA). miRNA cDNA samples were prepared by standard reverse transcription (RT) and preamplification procedures, as previously reported [64 (link)]. Preamplified samples were stored at −20 °C until expression analysis with the OpenArray system (Life Technologies) performed into 384-well OpenArray plates, according to manufacturer’s instructions. For each amplification curve, an AmpScore value was obtained. miRNAs with AmpScore < 1.1 or missing and Ct values > 27 were considered unamplified. The following assays (Life Technologies) were considered: hsa-miR-23a-3p 000399; hsa-miR-221-3p 000524; hsa-miR-423-5p 002340; hsa-miR-16-5p 000391; hsa-miR-26a-5p 000405; hsa-miR-103a-3p 000439; hsa-miR-101-3p 002253; hsa-let-7a-5p 000377; hsa-miR-425-5p 001516; U6 snRNA 001973; hsa-miR-146a-5p 000468; ath-miR159a 000338.
4
OXTR Genetic Risk Score Calculation
5
Genotyping of TCF7L2 rs7901695
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We genotyped for the TCF7L2 SNP in rs7901695. The DNA was extracted from peripheral blood leukocytes, by a salting-out method described previously in detail [19 (link)]. Genotyping for the SNP was conducted using TaqMan SNP technology with a ready-to-use human assay library (Applied Biosystems, Waltham, MA, USA), using an OpenArray System (Life Technologies, Grand Island, NY, USA) as a tool for high-throughput genotyping. The genetic analyses were performed in duplicate in accordance with the manufacturer’s instructions. To detect possible false-positive signals caused by contamination, a negative control consisting of a sample without a template was used.
6
Extracellular miRNA Profiling Protocol
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Five milliliters of cleared secretomes were 1:1 diluted in PBS and ultracentrifugated (100,000× g, 9 h at 4 °C). After addition of exogenous Arabidopsis thaliana ath-miR-159a (30 pg) synthetic miRNA spike to evaluate RNA recovery and cDNA synthesis, total RNA was extracted with miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany), as previously reported [93 (link)]. The expression of 754 miRNAs was evaluated with the OpenArray system (Life Technologies, Foster City, CA, USA) with 384-well OpenArray plates, according to the manufacturer’s instructions. Each single miRNA was considered as present and considered for further analyses only when amplification appeared in all three samples. ath-miR-159 spike-in CRT was used for the equalization of technical differences. Eventually, the global mean method [93 (link)] allowed the normalization between samples.
7
RNA Extraction and miRNA Expression Analysis
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miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany) were used to extract total RNA from similar iASC-EV numbers (26 ± 4 E9, mean ± SEM), following the manufacturer’s instruction. To monitor the process of RNA recovery and cDNA synthesis, before RNA extraction samples were spiked-in with exogenous ath-miR-159a (30 pg), an Arabidopsis thaliana synthetic miRNA. Specific primers for ath-miR-159a were provided in the RT and PreAmp primer pools (Life Technologies, Foster City, CA, USA). cDNA samples were prepared by standard reverse transcription (RT) and pre-amplification procedures, as previously reported [34 (link)]. Expression analysis with the OpenArray system (Life Technologies) was performed into 384-well OpenArray plates, according to the manufacturer’s instructions. An AmpScore value was obtained for each amplification and candidates with AmpScore <1.1 or missing, together with Crt values >25 were considered unamplified. When unamplified, an arbitrary Crt value of 25 was assigned. All assays for RGs and OA-miRNAs were purchased from Life Technologies.
8
Genetic Profiling: DNA Extraction and Genotyping
9
Profiling Serum miRNAs by qRT-PCR
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Serum miRNAs were profiled by qRT–PCR using the TaqMan OpenArray assays (Applied Biosystems). Briefly, 0.9–2.7 ng of isolated RNA was reverse transcribed in a 7.5 μl reaction volume using the TaqMan MicroRNA Reverse Transcription Kit and Megaplex RT Primers Human Pools A (v2.1) and B (v3.0) according to the manufacturer’s protocol (Applied Biosystems). The resulting cDNA (2.5 μl) was preamplified to increase the quantity of specific cDNA targets with Megaplex PreAmp Primers, Human Pools A (v2.1) and B (v3.0) and the TaqMan PreAmp Master Mix in a final volume of 25 μl (Applied Biosystems). The preamplified product was diluted 40 folds and mixed 1:1v/v with the TaqMan OpenArray Real-Time PCR Master Mix. 5 μl aliquots of the mixture were dispensed on an OpenArray 384-well sample plate. Subsequently, TaqMan OpenArray Human MicroRNA Panels were loaded with the OpenArray AccuFill System and the PCR reactions were carried out with an OpenArray System (Applied Biosystems) following the manufacturer's instructions. Values of Ct were processed and exported to DataAssist Software (Applied Biosystems).
10
Validating Imputation Accuracy via Genotyping
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To validate imputation accuracy, the imputed variant chr9:14344410:I (rs71321981) at 9p22.3 was genotyped by direct sequencing using standard methods as described elsewhere [69 (link)]. The chromatograms were analyzed manually, and the corresponding nucleotide sequences were compared to the reference sequence at 1000 Genomes browser (1000 Genomes Project; see URLs). The primer sequences are available from the authors on request.
In addition, imputed variants rs190200374 and rs80035109 at 15q21.2 were genotyped using a TaqMan® chemistry-based PCR platform (Open Array™ system) and custom-made TaqMan® SNP Genotyping assays (Applied Biosystems). The allelic calling analysis was performed using TaqMan Genotyper v1.3 software and OpenArray™ SNP Genotyping Analysing software. For quality control, two independent readers interpreted the results. Random selection of all samples (about 5% in H2000 and 12% in YFS) was re-genotyped. No discrepancies were discovered in the replicate tests for the variants.
Concordances between the genotyped and imputed SNPs were calculated using threshold 0.7 for converting probabilistic genotypes of imputed SNPs to hard calls.
In addition, imputed variants rs190200374 and rs80035109 at 15q21.2 were genotyped using a TaqMan® chemistry-based PCR platform (Open Array™ system) and custom-made TaqMan® SNP Genotyping assays (Applied Biosystems). The allelic calling analysis was performed using TaqMan Genotyper v1.3 software and OpenArray™ SNP Genotyping Analysing software. For quality control, two independent readers interpreted the results. Random selection of all samples (about 5% in H2000 and 12% in YFS) was re-genotyped. No discrepancies were discovered in the replicate tests for the variants.
Concordances between the genotyped and imputed SNPs were calculated using threshold 0.7 for converting probabilistic genotypes of imputed SNPs to hard calls.
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