Taqman preamp master mix 2x

Expression profiling of 188 miRNAs identified as the most consistent and reliable miRNAs in human plasma^{14 (link)} was performed using TaqMan-Low-Density-Array (TLDA) human custom arrays as instructed by the manufacturer (Applied Biosystem, CA, USA) with Applied Biosystems QuantStudio 7. Briefly, a fixed volume of 3 μL of miRNA solution was used for reverse transcription using a TaqMan MicroRNA Reverse Transcription Kit and the TaqMan Custom RT pool (Applied Biosystems, CA, USA), which were customized for TLDAs. The reaction was performed under the following conditions: incubation for 30 min at 16 °C and 30 min at 42 °C, inactivation for 5 min at 85 °C and immediate cooling to 4 °C. The cDNA was stored at − 20 °C. For our custom selection, 3.125 μL of the reverse transcription reaction was used for preamplification using TaqMan PreAmp Master Mix (2X) and TaqMan PreAmp pool (Applied Biosystems, CA, USA). The reaction conditions were as follows: 10 min at 95 °C, 2 min at 55 °C, 2 min at 72 °C, 12 cycles of 15 s at 95 °C and 4 min at 60 °C, inactivation for 10 min at 99.9 °C and cooling to 4 °C. The preamplification reaction was then diluted with water (1:3), and 4.5 μL was used for TLDA. Reverse transcription and preamplification reactions were carried out with an Applied Biosystems Verity Thermal Cycler. Data were normalized by the mean-centre normalization method.

TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany) was used for reverse transcription (RT). The 7.5 μl-reverse transcription reaction contained 0.8 μl Megaplex RT primers, 0.2 μl 100 mM dNTP, 1.5 μl Multiscribe Reverse Transcriptase, 50 U/μl, 0.8 μl RT-buffer, 0.9 μl MgCl₂ (25 mM), 0.1 μl Rnase inhibitor, 0.2 μl Nuclease free water and 3 μl extracted miRNA sample. Pre-amplification of converted DNA (cDNA) was performed with TaqMan PreAmp Master Mix (2X) (Applied Biosystems) and Megaplex PreAmp Primers (10X) (Applied Biosystems) using the Gene Amp PCR System 9700. The reaction volume of 25 μl consisted of: 12.5 μl TaqMan PreAmp Master Mix, 2.5 μl Megaplex PreAmp Primers, 7.5 μl nuclease-free water and 2.5 μl of the reverse transcript product. The pre-amplification was run on Gene Amp PCR System 9700.

HLSC and FIBRO EVs, derived from three different preparations, were used for the extraction of RNA by a mirVana RNA isolation kit (Ambion), according to the manufacturer’s protocol. miRNA content was analyzed using the qRT-PCR method and the Applied Biosystems TaqMan™ Array Human MicroRNA A + B Cards Set v3.0 (Applied Biosystems), run on QuantStudio 12K Flex (Applied Biosystems). Briefly, 140 nanograms of RNA were reverse transcribed with a Megaplex RT Pools kit (Applied Biosystems), according to the manufacturer’s protocol. TaqMan® PreAmp Master Mix 2X (Applied Biosystems) and specific Megaplex™ PreAmp Primers (10X) (Applied Biosystems) were used to pre-amplify each cDNA sample, which were then loaded onto the TaqMan MicroRNA Array. Raw Ct values, the automatic baseline, threshold and comparison of miRNA expression were analyzed using Expression Suite software (Thermo Fisher Scientific, Waltham, MA). Data were matched with the MSC EV miRNA expression dataset, published in Collino et al.^{30 (link)}.
Pathway enrichment analysis was performed using Funrich V3.1.3 software^{63 (link)}, and data with an adjusted p-value < 0.05 were considered to be statistically significant (Hypergeometric test with Boferroni correction).

TaqMan MicroRNA Reverse Transcription Kit (Life technologies, CA, USA) was used for 3 μl of extracted miRNAs. Pre-amplification of converted DNA (cDNA) was performed with TaqMan PreAmp Master Mix (2X) (Life technologies, CA, USA) and Megaplex PreAmp Primers (10X) (Life technologies, CA, USA). RT and pre-amplification were run on ProFlex™ PCR System (Life technologies, CA, USA). RT-qPCR for mature miRNAs was performed using 1 μl of the pre-amplificated cDNA product. Each miRNA was run in duplicate using the 7900HT Fast Real-Time PCR System from Applied Biosystems, MS,USA.

Primers and probes for miR-16, miR-215, miR-494, miR-4428, and RNU48 were synthesized by Life Technologies for reverse-transcription, cDNA pre-amplification, and quantitative real time reverse transcription-polymerase chain reaction (qPCR). cDNA was prepared from 100 ng of total RNA. To synthesize cDNA, the TaqMan miRNA reverse transcriptase kit (Life Technologies) was used. RT-PCR protocol was 16° C for 30 mins, 42° C for 30 mins, 85° C for 5 mins, and a final hold at 4C. cDNA preamplification was performed using TaqMan PreAmp Master Mix (2x) (Life Technologies). Preamplification protocol was 95° C for 10 min, 55° C for 2 min, and then 72° C for 2 min; following this, there were 12 cycles of 95° C for 15 sec followed by 60° C for 4 mins. Once cycling was complete, samples were held at 99.9° C for 10 mins and then at 4° C for a final hold. qPCR was performed with the Roche 480 Light Cycler (Roche, Indianapolis, IN) using the TaqMan Universal Master Mix II (Life Technologies); qPCR thermal profile included 95° C for 10 min followed by 45 cycles of 95° C for 15s, 60° C for 60s, and 72° C for 1s and a final hold of 40° C for 30s.

Total RNA (1 μg/50 μL) was reversed-transcribed into cDNA using random primers, deoxyribonucleotide triphosphates (dNTPs), RNAse inhibitor, and multiScribe RT enzyme using a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific), with the following heating cycles: 25°C for 10 min, 37°C for 2 h, and 85°C for 5 min. Assuming a retrotranscription efficiency of 100% and a final volume of 100 μL, the cDNA concentration was estimated to be 10 ng/μL.
Specific target preamplification of 1.25 μL cDNA was carried out with a mixture of 1.25 μL TaqMan™ Gene Expression Assay (0.2X, Thermo Fisher Scientific, MA, USA) and 2.5 μL TaqMan Preamp Master Mix 2X (Thermo Fisher Scientific), at 95°C for 10 min, followed by 15 PCR cycles, at 95°C for 15 s and 60°C for 4 min. The preamplification product was diluted 1 : 5 in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0; Teknova, Hollister, CA, USA).

A preamplification reaction was performed after the reverse transcription reaction on the cDNA samples using the TaqMan PreAmp Master Mix 2X (Thermo Fisher, Waltham, MA, USA) and a Primer Pool consisting of 0.2X (final concentration) of each TaqMan assay.
The reactions were performed in a 96-well cycler (Veriti 96-well cycler, Thermo Fisher, Waltham, MA, USA), according to the manufacturer’s protocol; enzyme activation at 95 °C for 600 s, followed by 14 cycles at 95 °C for 15 s and 60 °C for 240 s.
The amplified cDNA was analyzed on a 192.24 Dynamic Array on the Biomark HD Real-Time PCR system (Fluidigm, South San Francisco, CA, USA) using the standard Gene Expression Protocol: hold at 95 °C for 60 s (polymerase activation), followed by 35 cycles at 96 °C for 5 s (strand denaturation) and 60 °C for 60 s (annealing). All samples were measured in technical duplicates. The 2−∆∆Ct method was used to calculate the relative gene transcription. The transcription levels of the different gene transcripts were normalized against the mouse-specific TaqMan house-keeping gene assays, Table 2. Hprt1 (ABI, Mm00446968_m1) and β-actin (Mm02619580_g1) were used as house-keeping genes.