Taqman array human microrna a cards v2

In the screening phase, 377 miRNAs were analyzed in 12 selected patients to identify miRNAs associated with response to neoadjuvant treatment. miRNA profiling was performed with TaqMan Array Human MicroRNA A Cards v2.0 (Applied Biosystems, ThermoFisher Scientifics) using pre-amplification as previously described[22 (link)]. Expression levels were calculated by the 2^-ΔΔCt method. Real-time quantitative PCR reactions were performed on an ABI 7900 HT Sequence Detection System (Applied Biosystems). Normalization was performed with RNU48, based on preliminary analyses comparing the stability of RNU48, RNU44 and MammU6; RNU48 had the lowest variability of expression in the miRNA expression patient data set and was therefore used in this study. All miRNAs that were expressed in less than 10% of samples or with an unreliable quantification were excluded from further analysis, leaving a set of 184 miRNAs.
In the validation phase, selected miRNAs identified in the screening phase were validated in the whole cohort of 96 patients by single Real Time TaqMan MicroRNA Assays in an Applied Biosystems 7500 Sequence Detection System as previously described[23 (link)].

Total RNA purified from the tissues of 18 patients was used for miRNA screening assay. The specimens of PDAC cohort were divided into four groups, i.e., 5 lymph node metastases positive (N+), 5 negative (N−), 5 grade G3, and 5 grade G2 tissue samples. For reverse transcription, an equal amount of RNA tissue from different patients was mixed into 4 pools according to two specific clinical–pathological features: lymph node metastasis (N+ versus N− group) and tumor grading (G3 group versus G2 group). N− group and G2 group were used as control groups. Starting from each RNA pool, cDNA was synthesized with Megaplex RT Primers, Human Pool A v2.1 (Applied Biosystems, Foster City, CA, USA), and TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, CA, USA), according to the manufacturer’s instructions. miRNA expression profiling by TaqMan Array Human MicroRNA A Cards v2.0 (Applied Biosystems, CA, USA) and the Ct value determination were performed as previously described [58 (link)].

CRC organoid conditioned media (1 mL) were harvested after 48 h, they were centrifuged at 300× g for 5 min, 2000× g for 20 min, and 12,500× g for 20 min. Forty µL anti-CD63 and 20 µL anti-CD81-coated beads were added to the supernatant after centrifugation, the samples were incubated for 16 h at +4 °C and were washed with PBS 3–4 times. EVs bound to beads were then lysed in Qiazol (Qiagen). Total RNA was isolated with the miRNEasy Micro Kit (Qiagen) following the manufacturer’s protocol.
Three µL total RNA was reverse transcribed with Megaplex RT primers, the samples were pre-amplified with Megaplex PreAmp Primers (Thermo Fisher) and the TaqMan™ Array Human MicroRNA A Cards v2.0 (Thermo Fisher) were measured on an ABI 7900HT instrument according to the manufacturer’s protocol and according to [23 (link)].

RNA was isolated and quantified from the discovery cohort of patients as described previously [34 (link)]. Briefly, RNA was isolated from 150 µL of plasma using Trizol liquid sample (LS) reagent (ThermoFisher, Waltham, MA, USA), then converted to cDNA using the High Capacity Reverse Transcription Kit with multiplexed reverse transcription (RT) primers (ThermoFisher). Synthesized cDNA was then pre-amplified using PreAmp MasterMix with multiplexed TaqMan (TM) primers corresponding to the RT primers used in initial cDNA reaction. Quantitative analysis of miRNA was performed via TaqMan Low-Density Array Cards (TaqMan™ Array Human MicroRNA A Cards v2.0; ThermoFisher). The ThermoFisher Cloud software suite with the Relative quantification (Rq) application was used to perform statistical analysis and determine expression of miRNA in either mild or severe FSHD1 patient groups versus healthy controls. A value > 1 indicates an increase and a value < 1 indicates a decrease in miRNA expression in FSHD1 versus healthy controls, with p-values ≤ 0.05 considered significant. To reduce false-positive discovery in this setting, we used an evidence-based approach where candidate miRNAs that significantly increased in the discovery groups were cross-referenced to a separate set of non-overlapping CINRG patients used as a validation group.