Taqman microrna expression assay

Real-time PCR (qPCR) validation of miRNAs was performed by using cDNA prepared from 10 ng of total RNA, enriched from uE, using commercially available Taqman micro-RNA expression assays (Applied Biosystems, Foster City, CA, USA) as per the manufacturer’s instructions. For mRNA, high-capacity cDNA synthesis kit (applied biosystems) was used for cDNA conversion according to the manufacture’s instruction. Takara TB Green Premix Ex Taq probes were used for relative quantification as per manufacture’s instruction. qPCR was performed using the BIO-RAD CFX96 Touch Real-Time PCR Detection System. All the reactions were performed in duplicates. Data were analyzed using the 2^-ΔCt method.

Reverse transcription for miRNAs was performed using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). TaqMan MicroRNA expression assays (Applied Biosystems) were used to provide specific primers for the reverse transcription and quantitation of mature miR-34a and RNU6B. Thermal cycling conditions for qPCR were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. RNU6B expression was used as an internal control for miR-34a expression. In order to detect mRNA, 500 ng of RNA was reverse-transcribed using a PrimeScript Reverse Transcriptase reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions, and was amplified by qPCR using specific primers (Table II). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control to normalize for differences in the input RNA. The PCR conditions were as follows: One cycle at 95°C for 1 min and 34 cycles at 94°C for 10 sec, 60°C for 30 sec and 72°C for 15 sec. All the measurements were performed in triplicate. Amplification was analyzed using the ΔΔCt method (24 (link)).

We used commercial TaqMan microRNA Expression Assays (Applied Biosystems, Foster City, CA, USA) probes against miRNA-142-3p, miRNA-146a, miRNA-186, miRNA-142-5p, miRNA-222, miRNA-181a, miRNA-502-3p and miRNA-26a. MiRNA expression of FFPE tissues was achieved using the Applied Biosystems 384-well multiplexed real-time PCR assay with 10 ng of total RNA. RNA from each case was reverse-transcribed using the TaqMan® MicroRNA Reverse Transcription kit (Applied Biosystems). miRNA-qRT-PCR was performed using TaqMan® MicroRNA Assay (Applied Biosystems). All reactions were run on the ABI PRISM HT 7900 Real-Time Sequence detection system (Applied Biosystems), in accordance with the manufacturer’s protocol. Two noncoding RNAs (RNU44 and RNU6B) were used as endogenous RNAs. Ct values were exported using Sequence Detection System version 2.2.2 software (Applied Biosystems) and the data were analyzed with Real Time StatMiner (Integromics). Reproducibility of triplicate curves was evaluated: inconsistent replicates were omitted. An miRNA was considered to be present if the Ct was less than 36 in all three biological replicates.

The miRNA and mRNA expression levels were analyzed by quantitative real-time PCR. The reactions were carried out on a StepOne™qPCR Real-Time PCR machine, containing 1X Master mix (Applied Biosystems^®), with 1X probes (TaqMan^® microRNA Expression Assays: hsa-miR-210-3p: TM000512, hsa-miR-218-1-3p: TM-002094, hsa-miR-221: TM-002096, hsa-miR-1233-3p: TM-002768 and TaqMan^® mRNA Expression Assays: CXCR4: Hs00607978_s1, Applied Biosystems^® ), cDNA sample (≈ 50 ng), RNU-48 endogenous control for miRNA normalization (TaqMan^® Gene Expression Assays, TM-001006, Applied Biosystems^®) and Human GUSB (Beta Glucoronidase) endogenous control (Applied Biosystems^®) for mRNA normalization.
The amplification conditions were as follows: holding stage 95°C for 20 seconds, followed by 45 cycles of 95°C for 1 second and 60°C for 20 seconds. Three technical replicates were made for each sample.
Data analysis was made using StepOne™Sofware v2.2 (Applied Biosystems^®) with the same baseline and threshold set for each plate, in order to generate quantification cycle (Cq) values for all the miRNAs in each sample.

Each reaction was performed with 5 µL of 2× TaqMan^® Fast Advanced Master Mix (Applied Biosystems^®) with 0.5 µL of 20× TaqMan^® MicroRNA Expression Assays (miR-466l-3p: 002804; miR-466k: 240990_mat; miR-223-3p: 002295; miR-125b-5p: 000449; snoRNA-202: 001232; Applied Biosystems^®), 3 µL of nuclease-free water and 1.5 µL of cDNA sample, making a total volume of 10 µL. The quantification was performed in duplicate, and CT standard deviation values superior to 0.5 were excluded. Negative controls lacking cDNA were also included in all reactions. All target and endogenous controls for each sample were amplified in the same plate. The thermal cycling conditions for all assays were the following: 20 s at 95 °C followed by 45 cycles of 1 s at 95 °C and 20 s at 60 °C. The same baseline and threshold were set for each plate using the analysis software for qPCR from the Thermo Fisher Connect platform (Thermo Fisher Scientific, Waltham, MA, USA), in order to generate threshold cycle (Ct) values for all of the miRs/SnoR in each sample. Small nucleolar RNA 202 (snoR-202) was previously tested by our group in this mouse model, was the one that showed the lowest standard deviation values in chest and ear, and therefore, was used as endogenous control [14 (link),15 (link)].

Mature miRNAs were assayed using the TaqMan microRNA expression assays (Applied Biosystems) in accordance with the manufacturer's instructions. TaqMan assays used were miR-28-3p (TM002446), miR-148a-3p (Tm000470) and U6 (TM001973). The comparative Ct method was used to calculate the relative changes in gene expression on the 7500 Fast Real Time PCR System.

To determine whether MSC-derived MVs cargo modifies the microRNAs expression of CD34⁺ cells, miR-10a and miR-15a were analyzed by RT-PCR. Total RNA was extracted from CD34⁺ cells that had been co-cultured with and without MVs (7 MDS and 5 controls) using Trizol reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. We performed individual quantitative PCR for the following microRNAs: Hsa-mir-10a (TM:000387), Hsa-mir-15a (TM:000389) and RNU43 (TM:001095, Applied Biosystems), the latter was used as control. cDNA was prepared to be retrotranscribed with a TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems) and the expression was quantified using commercial TaqMan^® MicroRNA Expression Assays and the Step One Plus Real-Time PCR System (Applied Biosystems). All samples were performed in duplicate. Relative quantification was calculated from the 2^−ΔCt values with the equation: ΔCt = Ct_microRNA - Ct_RNU43. Results were expressed as the ratio between CD34⁺ cells with MVs from MDS or HD and the same CD34⁺ cells without MVs. To evaluate which metabolic pathways are regulated by these two microRNAs DianaLab miR Path web was used.