Reverse transcription master mix kit

Total RNA was extracted from pre-weighed -80 ^oC frozen LD and ST tissue using peqGOLD TriFast (VWR International GmbH, Hannover, Germany), purified using RNeasy minikits (Qiagen, Hilden, Germany) and quantified using a Nanophotometer (Implen GmbH, Munich, Germany). The RNA quality was assessed using a Bioanalyzer 2100 and RNA 6000 Nano kit (Agilent Technologies, Waldbronn, Germany), with a RIN cutoff of 8.5. Synthesis of cDNA was performed using 250 ng of purified RNA and the reverse transcription master mix kit (Fluidigm, Amsterdam, Netherlands), according to the manufacturer’s instructions.

cDNA was generated by subjecting 50 ng of RNA from each sample to reverse transcriptase reaction (Reverse Transcription Master Mix Kit Fluidigm P/N-100-6297). Then, 1.25 µL of each cDNA solution was used to generate a preamp mix containing a pool of the 26 primers pairs and the PreAmp Master Mix Kit (Fluidigm P/N-100-5744). Preamp mixes were run for 14 cycles and the remaining primers were digested with Exonuclease I (New England BIOLAB. P/N M0293l. LOT 0191410). Preamp samples were analyzed for the expression of 22 genes of interest (for primer sequences, see Supplementary Table 3) using the BioMark qPCR platform (Fluidigm, San Francisco, CA, USA). Data were normalized to Gadph, B2m, Actb and Gusb (from the same animal) and fold changes were calculated using the 2-ΔΔCt method⁹⁴ .

The donor-matched VB-BM and IC-BM MSCs were cultured for 1, 2 and 3 weeks in either osteogenic or chondrogenic media as described above. The RNA isolation was performed using Single Cell RNA purification Kit (Geneflow Ltd, Lichfield, UK). The genomic DNA was eliminated from RNA samples using RNase-Free DNase I Kit (Geneflow), and then cDNA was produced using reverse transcription master mix kit (FLUIDIGM, UK). The TaqMan probes (all from Thermo Fisher Scientific) were used to measure the genes of the osteogenic transcription factors; Osteonectin (SPARC) and Runt-related transcription factor 1 (RUNX2), Osteopontin (SPP1), alkaline phosphatase (ALP), Collagen type I alpha 2 (COL1A2) and Osteocalcin/bone gamma-carboxyglutamic acid-containing protein (BGLAP) as well as genes related to chondrogenesis; aggrecan (ACAN), Collagen type 2 (COL2) and SRY-Box 9 (SOX9). The real time PCR assays were run on QuantStudio™ 7 Flex Real-Time PCR System, 384-well (Thermo Fisher Scientific). As published before [40 (link)], the data were analysed relative to the housekeeping gene, hypoxanthine-guanine phosphoribosyltrans- ferase (HPRT1), then the fold change of gene in differentiated cells was calculated relative to undifferentiated cells.