Expression suite software v1

Reverse transcription of purified miRNAs was performed in accordance with the protocol of a universal TaqMan™ Advanced miRNA cDNA Synthesis Kit (Applied Biosystems). Expressions of individual miRNAs were determined via a quantitative RT-PCR method using TaqMan™ Advanced miRNA Assays and TaqMan™ Fast Advanced Master Mix (Applied Biosystems). The plates with assays were prepared using the automated liquid handling system epMotion 5075 (Eppendorf). All reactions were run in triplicates on the QuantStudio 12K Flex Real-time PCR System. Results were processed in ExpressionSuite Software v1.0.3 (Applied Biosystems™, Carlsbad, CA, USA), qBase+© v2.4 (Biogazelle, Gent, Belgium) and Statistica software version 12 (StatSoft, Inc., Tulsa, OK, USA). Comparison of the miRNAs’ expressions between patients and controls was performed with Mann–Whitney’s U-test with the Benjamini–Hochberg correction to multiple testing (p-value ≤ 0.05; fold change ≥ 2).

Total RNA was extracted from MDA-MB-231 with Quiazol reagent (QIAGEN). We used the miRNeasy kit to extract small RNAs and mRNA following the manufacturer's instructions, and miRNAs were quantified using a fluorometer Qubit 4 (Thermo Fisher Scientific Inc). For miRNA RT-qPCR, cDNA from total RNA was generated with the miScript II RT Kit (QIAGEN).
For mRNA RT-qPCR, cDNA was synthesized with 1 μg of total RNA using iScriptTM cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions in a total volume of 20 μl. Transcript of cDNA was analyzed in triplicate by RT-qPCR performed with Applied Biosystems ViiA 7 7900HT thermocycler using iTaq Universal SYBR Green Supermix (Bio-Rad). Relative quantification analyses were performed using the software SDS2.2 and Expression Suite Software V1.0.3 (Applied Biosystems, Thermo Fisher Scientific Inc), online software ThermoFisher Cloud and QuantStudio RT-qPCR Software (Thermo Fisher Scientific Inc), and Excel.
Fold changes of miRNA and gene expression were calculated relatively to housekeeping expression, 18S for gene and Snord95 for miRNA, using the comparative cycle threshold CT (ΔΔCT) method.

For the discovery phase, we determined the serum miRNA expression profile of three RNA pools (five samples per pool) of each study group using the TaqMan Human MicroRNA Array Panel v3.0 (Applied Biosystems, CA, USA), which includes cards A and B in a 384-well format and probes for 754 human miRNAs. The quantification procedure was performed according to the manufacturer’s instructions. Briefly, 30 ng of pooled RNA was reverse transcribed (RT) using the Megaplex RT stem-loop primer pools A and B (Applied Biosystems, Foster City, CA, USA). Subsequently, Megaplex RT products were pre-amplified using Megaplex PreAmp Primers (Pool A and B) and TaqMan PreAmp Master Mix (Applied Biosystems). Real-time PCR reactions were performed on the 7900 HT (Applied Biosystems) with the recommended cycling conditions. The differential expression of miRNAs between study groups was assessed using the Expression Suite Software v1.0.3 (Life Technologies). Data were normalized using the mean of the expression value (Ct) of all expressed miRNAs on the plate [20 (link)]. Only Raw Ct values lower than 35 were considered for analysis. The levels of miRNAs that showed values of p < 0.05 and a log2 foldchange > 2 in AD patients compared with those in controls were considered differentially expressed.

For real-time quantitative PCR (RT-qPCR), hearts were harvested, washed to remove blood clots, weighed, and frozen in RNAlater (Life Technologies, USA). Total RNA (for gene expression studies) and DNA (for parasite detection/quantification) were extracted from the same sample by using Tri reagent (Sigma-Aldrich, USA), according to the manufacturer's instructions. All reverse transcriptase reactions were performed by using a SuperScript III first-strand synthesis kit, and RT-qPCR was performed by using TaqMan gene expression assays for TNF (catalog number Mm00443258-m1) and the endogenous housekeeping control genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number Mm99999915-g1) and β-actin (catalog number Mm00607939-s1), which were purchased from Life Technologies. Reactions were performed in duplicate according to the manufacturer's instructions, using a cDNA template obtained from 2 μg RNA. The conditions for PCR were as follows: 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Relative quantification of target gene levels was performed by using the comparative threshold cycle (C_T) (ΔΔC_T) method (23 (link)). RT-qPCR data were normalized by mRNA levels of the housekeeping genes GAPDH and β-actin by using Expression Suite software V1.0.3 (Life Technologies, USA), and fold increases were determined by comparison with NI controls.

To evaluate the post-transfection miR-320a expression levels in hOBs, total RNA was extracted 48 hours after transfection using the miRNeasy mini kit (Qiagen) according to manufacturer instructions. Then, 1 μg of total RNA was reverse-transcribed in 20 μl reactions using the miScript II RT kit (Qiagen). cDNA was diluted 1/8 and 2 μl were assayed in 10 μl qPCR reactions in 384-well plates using MiScript SYBR Green PCR kit according to the protocol. The mature miR-320a sequence, according to the mirBase web site, was used as a forward primer (5’-AAAAGCTGGGTTGAGAGGGCGA-3’) and the Universal primer as a reverse. U6 snRNA was used as the reference gene for normalization. All qPCR reactions for each sample were performed in triplicate. Amplification was performed in a QuantStudio 12K Flex Real-Time PCR (Applied Biosystems), and the ExpressionSuite software v.1.0.3 (Life Technologies) was used both for determination of relative quantification (RQ) (by 2-ΔΔCt method) and for melting curve analysis.

Complementary DNA was synthesized from kidney cortex total RNA (200 ng) using TaqMan MicroRNA Reverse Transcription kit (Life Technologies) and Megaplex^™ TLDA A v2.1 and TLDA B v3.0 RT Primers. Resulting cDNA was used for qPCR. Quantitative real-time PCR was performed using TaqMan Universal Master Mix II (Life Technologies) and TaqMan TLDA Human MicroRNA A version 2.0 and TLDA Human MicroRNA B version 3.0 cards. Sample loaded TLDA cards were run on QuantStudio 12K Flex real-time PCR system (Life Technologies). Generated raw data files were analyzed using Expression Suite Software v1.0.3 (Life Technologies).