Telotaggg telomere length assay kit

Telomere lengths were determined by Southern blot using a TeloTAGGG Telomere Length Assay Kit (Roche) according to the manufacturer’s protocol. Briefly, 1 µg of each DNA sample was digested with RsaI and HinfI for overnight at 37 °C, electrophoresed on a 0.8% agarose gel at 50 V for 4 h, and transferred to a nylon membrane by Southern blotting. The membrane was blocked and hybridized overnight to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. Then, it was incubated with anti-DIG-alkaline phosphatase (1:1000 dilution) for 30 min and processed using the substrate in the TeloTAGGG Telomere Length Assay Kit (Roche). After chemiluminescence signals were visualized with a ChemiDoc XRS system (Bio-Rad), telomere length was calculated with Telo Tool version 1.3.

Telomere length was determined by Southern blotting (TRF analysis) using a TeloTAGGG Telomere Length Assay Kit (Roche) according to the manufacturer’s protocol. Briefly, 1 μg of each DNA sample was digested with Rsa I and Hinf I overnight at 37 °C, electrophoresed on a 0.8% agarose gel at 50 V for 4 h and then transferred to a nylon membrane by Southern blotting. The blotting membrane was blocked and hybridized overnight to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. The washed blot was incubated with anti-DIG-alkaline phosphatase (1:1000 dilution) for 30 min and developed using the substrate in the TeloTAGGG Telomere Length Assay Kit (Roche). The chemiluminescence signals were visualized with a ChemiDoc XRS system (Bio-Rad), and TRF analysis was performed with TeloTool version 1.3 [47 (link)].

Telomere restriction fragments were analyzed using the TeloTAGGG Telomere Length Assay kit (Roche). Briefly, cells were harvested by trypsinization, washed in PBS and collected by centrifugation at 400 g for 4 min. Genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen), digested with HinfI and RsaI restriction enzymes (New England Biolabs) and separated by gel electrophoresis either on 0.8% agarose gels at 50V overnight in 1X TBE buffer or (to resolve elongated telomeres at later time points) on 1% megabase agarose gels (Bio-Rad) using a CHEF DRII equipment (Bio-Rad) under the following conditions: 120° field angle, 5 to 30 s switch times, 5 V/cm and 14°C for 14 hr in 1X TAE. Following the resolution of DNA fragments, DNA was transferred to a positively charged nylon membrane (Roche) by Southern blotting and hybridized with a digoxigenin-labelled telomeric probe. Membranes were exposed to X-ray film (Carestream) and developed in X-OMAT 2000 Processor (Kodak). Mean telomere lengths were calculated as described in Kimura et al. (2010) (link).

The TeSLA procedure was carried out as described in Lai et al. (2017) (link), with minor modifications. Oligonucleotide sequences for the ligation and amplification reactions were published in Lai et al. (2017) (link).
Briefly, 50 ng of genomic DNA was ligated with TeSLA-T oligonucleotides and then digested with CviAII, BfaI, NdeI, and MseI restriction enzymes (New England Biolabs), followed by shrimp alkaline phosphatase (New England Biolabs) treatment. The digested DNA was ligated with double-stranded TeSLA adapters, and 30 pg of the ligated DNA was subsequently used for long-range PCR amplifications. Amplification reactions were carried out in 25 μl volume, using 2.5 units of FailSafe Enzyme Mix (Lucigen) with FailSafe buffer H and 0.25 μM primers (AP and TeSLA-TP). After the initial melt at 94°C for 2 min, 25 PCR cycles were carried out (94°C for 15 s, 60°C for 30 s, and 72°C for 15 min). Amplified PCR products were resolved on a 1.2% agarose gel at 50V overnight in 1X TBE buffer. Southern blotting and telomere signal detection was performed using the TeloTAGGG Telomere Length Assay kit (Roche), as described for the TRF assay. The TeSLA-QUANT software was used for image quantification and statistical analysis, as described in Lai et al. (2017) (link).

Telomere length was determined by flow-FISH methodology by using the Telomere PNA kit/FITC from Dako (Glostrup, Denmark) according manufacture's introduction.
Additionally, telomere length was analysed by telomere restriction fragment (TRF) Southern blot by using the TeloTAGGG Telomere Length Assay Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Mean telomere length was determined by densitometric analysis of autoradiographies by using the Telometric© 1.2 software (Biostatistics and Bioinformatics Facility, Fox Chase Cancer Center, Philadelphia, PA, USA).

Telomere restriction fragment (TRF) analysis was performed with the TeloTAGGG Telomere Length Assay kit from Roche according to manufacturer’s instructions.