Total nucleic isolation kit

Tert, Terf-1, and Terf-2 gene expression was analyzed in RNA extracts of ventricular tissue. 10 mg of tissue were homogenized in 300 μl Magna Pure Lysis Buffer (Roche, Vienna, Austria) using the MagnaLyser (Roche, Vienna, Austria). RNA was extracted from these homogenates with the Total Nucleic Isolation Kit (Roche, Vienna, Austria) on a MagNA Pure LC instrument (Roche, Vienna, Austria). Subsequently, the mRNA in these extracts was transcribed into cDNA using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Finally, mRNA expression of TERT, TERF-1, and TERF-2 was analyzed by qPCR with TaqMan probes (Life Technologies dba Invitrogen, Waltham, MA, USA). The expression of each target gene was calculated with the ΔΔCT method using β-actin as a reference gene. The sequences of the probes used are listed below:

Approximately 10 mg derived from ventricular tissue were homogenized in 300μl Magna Pure Lysis Buffer (Roche, Vienna, Austria) using the MagnaLyser (Roche, Vienna, Austria). DNA was isolated with the MagNA Pure LC instrument (Roche, Vienna, Austria) using the Total Nucleic Isolation Kit (Roche, Vienna, Austria). Subsequently, relative telomere length (RTL) of peripheral blood mononuclear cells (PBMCs) was measured by quantitative real-time PCR (qPCR) using a protocol developed by Cawthon (63 (link)). This assay quantifies the ratio of average TL (T) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single-copy reference gene (S). The single-copy gene is used as an amplification control for each sample and to determine the number of genome copies per sample. All qPCR analyses were performed on Thermocycler CFX384 TouchTM (Biorad, Feldkirchen, Germany) using the following primers:
All primers have been purchased from Eurofins Genomics, Austria. Each run included a standard curve made by dilutions of isolated and pooled rat DNA from 21 different blood samples, to determine the quantity of the targeted templates. RTL has been calculated as the ratio of telomere quantity to single-copy reference gene quantity (T/S ratio).

TERT, TERF-1, and TERF-2 gene expression was analysed in RNA extracts of all solid organs. As blood leucocytes were used up for the measurement of RTL, they were not available for mRNA expression analyses. Therefore, mRNA expression in spleen was used as reference because the organ belongs to the lymphatic system and is rich in leucocytes. From each organ, 10 mg of tissue were homogenised in 300 µL Magna Pure Lysis Buffer (Roche, Wien, Austria) using the MagnaLyser (Roche, Wien, Austria). RNA was extracted from these homogenates with the Total Nucleic Isolation Kit (Roche, Wien, Austria) on a MagNA Pure LC instrument (Roche, Wien, Austria). Subsequently, the mRNA in these extracts was transcribed into cDNA using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Finally, mRNA expression of TERT, TERF-1, and TERF-2 was analysed by qPCR with TaqMan probes (Life Technologies dba Invitrogen, Waltham, MA, USA). The expression of each target gene expression was calculated with the ΔΔCT method using β-actin as reference gene. The sequences of the probes used were as follows:5.

Β-actin: 5′-CTTCCTTCCTGGGTATGGAATCCTG-3′;

6.

Tert: 5′-ATCGAGCAGAGCATCTCCATGAATG-3′;

7.

Terf-1: 5′-AAAACAGACATGGCTTTGGGAAGAA-3′;

8.

Terf-2: 5′-GAGAAAATTTAGACTGTTCCTTTGA-3′.