Telotaggg kit

DNA was extracted from MC‐MSC, WJ‐MSC and BM‐MSC every third passage using the High Pure PCR Template Preparation Kit (Roche, Sussex, UK) and stored at −20 °C until needed. DNA content was measured using a NanoDrop (Fisher Scientific, Loughborough, UK). The TeloTAGGG kit (Roche) was used to determine the length of telomeres from MC (n = 2), WJ (n = 2) and BM‐MSC (n = 1) over several passages according to the manufacturer's instructions. Genomic DNA (1 μg) from each sample population was digested with a HinfI/RsaI mixture for 2 h at 37 °C and then loaded onto a 0.8% agarose gel. The DNA fragments were separated by gel electrophoresis for 2–4 h at 70 V and transferred to a nylon membrane (Macherey‐Nagel, Düren, Germany) by Southern blotting.
The blotted DNA fragments were hybridized to a digoxigenin (DIG)‐labelled probe specific for telomeric repeats and incubated with a DIG‐specific antibody covalently coupled to alkaline phosphatase, which was visualized by the chemiluminescence substrate CDP‐Star. The telomere bands were then demonstrated by exposing the blot to an X‐ray film at room temperature for 15–20 min and the average terminal restriction fragment (TRF) length was determined by comparing the signals relative to the molecular weight standard.

Telomere length assays were performed using the teloTAGGG kit (Roche Diagnostics Ltd., West Sussex, UK). One microgram of genomic DNA was digested with HinfI/RsaI. Digests were separated by gel electrophoresis and blotted onto positively charged membrane (Roche Diagnostics Ltd., West Sussex, UK). Membranes were UV cross-linked, baked at 120 °C and washed in 2×SSC solution. Hybridization of DIG-labeled telomeric probe was performed using buffers and probe provided. Membranes were washed, probed with alkaline phosphatase-conjugated anti-DIG and exposed to CDP-star. Experiments were performed twice.

Telomerase activity, ALT status by telomere restriction fragment analysis and mRNA levels of hTERT and TERC expression were analyzed as previously described.^{18 (link)} To confirm ALT status, C-circle assay was performed based on previously described methods.^{28 (link)} Briefly, following φ29 amplification, DNA was transferred to a charged nylon membrane and detection was carried out using the TeloTAGGG kit (Roche Diagnostics, Indianapolis, IN) following manufacturer’s instructions for telomere detection. Samples showing positive signal were considered ALT positive while those with no signal were ALT negative. For hTERT promoter mutations (C228T and C250T), DNA was extracted as previously described.^{18 (link)} A Region of the hTERT promoter harboring the recurrent mutations C228T and C250T was amplified using forward (5ʹ-AGCACCTCGCGGTAGTGG-3ʹ) and reverse (5ʹ-GTCCTGCCCCTTCACCTT-3ʹ) primers followed by Sanger sequencing of the amplified product.