Taqman gene expression assay

For in vitro samples, the cells in culture were rinsed with PBS, scraped and the collected pellets were frozen at −80 °C until processing. For in vivo samples, excised, cell-containing constructs were collected and frozen at −80 °C until processing.
Quantitative RT-PCR were performed using the TaqMan® gene expression assay and an iCycler thermocycling apparatus (MyiQ^TM, Bio-Rad). All reagents were purchased from Applied Biosystems. Each RT-PCR analysis was performed in triplicate using 25 ng cDNA each time.
To identify the origin of cells in the implanted constructs, specific amplifications of the human GAPDH and human/mice 18S cDNA were performed using RT-PCR. No cross-reactivity between human and murine cDNA was observed when amplification of human GAPDH was performed using the specific TaqMan® gene expression assay.
Extracted mRNAs were analyzed using RT-PCR targeting murine osteogenic differentiation markers, specifically, Runt-related transcription factor-2 (RunX2), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), Osteonectin (ON) and Osteocalcin (OC) and human osteogenic differentiation markers, specifically, RunX2, ALP, BSP, OC, BMP-2, BMP-4 and BMP-6. The sequences of oligonucleotide primers used for the real-time PCR are listed in the Supplemental Data section, Table S1.

Digital droplet PCR was conducted to quantify the concentration of the R transcripts in prostate cancer cells and skin fibroblasts. TaqMan Gene Expression Assay was used to confirm the PTBP3 gene expression profile by another quantifying method: ddPCR using the QX100 system (Bio-Rad, Hercules, CA, USA). The copy numbers for each target were averaged across duplicates and normalized to PBGD references genes. Endogenous mRNA levels were measured by ddPCR using the QuantaLife droplet digital PCR system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Briefly, the 20-μL ddPCR mixture was prepared by combining cDNA, primers and probes with ddPCR Supermix (Bio-Rad, Hercules, CA, USA). Oil emulsion droplets for each sample were prepared using the QuantaLife droplet generator (Bio-Rad, Hercules, CA, USA). PCR reaction was under the following conditions: One cycle at 95 °C for 10 min, 45 cycles of 95 °C for 30 s, 60 °C for 1 min, and 98 °C for 10 min in a standard thermal cycler (Bio-Rad, Hercules, CA, USA). Plates were read on a QuantaLife droplet reader (Bio-Rad, Hercules, CA, USA), and the concentrations (copy numbers) of the targets in the samples were determined using the QuantaSoft software (QuantaSoft Analysis Pro software version 1.0).

After 1 day in culture in differentiation medium, SOD1^G93A and WT cells were lysed with TRIZOL^® reagent (Life Technologies). Total RNA was extracted using Direct-zol™ RNA Micro-Prep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. RNA was then pre-treated with RQ1 DNase (Promega, Milan, Italy) for eliminating genomic DNA contamination. Retrotranscription of 400 ng RNA was performed with SensiFAST™ cDNA synthesis kit (Bioline, London, UK). For real-time PCR, several mixes were prepared according to the number of interested genes. Each mix included Master Mix 2x (Life techonologies), 250 nM probe (for GPR17 Mm02619401_s1, for Rpl13a Mm05910660_g1) and 20 ng of cDNA. Gene-expression was analyzed with TaqMan^® Gene Expression Assay and normalized to housekeeping gene Rpl13a expression using CFX96 real-time PCR system (BioRad Laboratories) following the manufacturer’s protocol.

Total RNA was extracted from 5 × 10⁶ cells using ISOLATE II RNA Mini Kit (Bioline Reagents Ltd., London, UK) and stored in TE buffer (5 mM Tris-HCl, 0.1 mM EDTA, and pH 8.5 in DEPC-treated water) at −20°C until further analysis. First-strand cDNA was synthesized from total RNA using High Capacity cDNA Reverse Transcription Kit. A sample of 2 ng of total RNA was used as a template in a total volume of 20 µL. Real-time quantitative PCR for murine NADH dehydrogenase subunit 3 (ND3, assay ID: Mm04225292_g1), murine cytochrome b (CYB, assay ID: Mm04225271_g1), and murine β-actin (Actb, assay ID: Mm00607939_s1) was performed using the TaqMan Gene Expression Assay in a thermal cycler CFX96 Real-Time PCR Detection System (BIO-RAD Laboratories, Hercules, CA, USA). The thermal cycling conditions were as follows: 10 min of polymerase activation at 95°C, followed by 40 cycles of 30 s denaturation at 95°C and 60 s annealing/extension at 60°C. Each sample was run in duplicate. The cycle threshold (Ct) values were calculated by CFX96 Real-Time PCR Detection System (BIO-RAD) software and the comparative Ct method (2^−ΔCt model) was used to calculate relative fold-changes in gene expression and normalized to the average of Actb.