Quantitative PCR

Total RNA was isolated using an RNeasy Micro extraction kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s protocol. An on-column DNase I digestion was performed to avoid genomic DNA amplification. RNA level and quality were checked using the Nanodrop technology. A total of 500 ng of RNA was used for reverse transcription using the Superscript III reverse transcription kit (Invitrogen). Q-PCR analysis was performed using an ABI 7900 system or a QuantStudio 12 K Flex real-time PCR system (Applied biosystem) and TaqMan gene expression Master Mix (Roche) or Luminaris Probe qPCR Master Mix (Thermo Scientific), respectively, following the manufacturers’ instructions. Quantification of gene expression was based on the DeltaCt Method and normalized to 18S expression. PCR primers were previously described by S. Rodriguez and colleagues⁴⁵. Primer sequences were lamin A (exons 11/12), 5′-TCTTCTGCCTCCAGTGTCACG-3′ and 5′-AGTTCTGGGGGCTCTGGGT-3′; lamin C (exons 9/10), 5′-CAACTCCACTGGGGAAGAAGTG-3′ and 5′-CGGCGGCTACCACTCAC-3′ and progerin (exons 11/12), 5′-ACTGCAGCAGCTCGGGG-3′ and 5′-TCTGGGGGCTCTGGGC-3′; ALPL, 5′-CCACGTCTTCACATTTGGTG-3′ and 5′-AGACTGCGCCTGGTAGTTGT-3′; COL1A1, 5′-CCCCTGGAAAGAATGGAGAT-3′ and 5′-CCATCCAAACCACTGAAACC-3′; OCN, 5′-GCTGAGTCCTGAGCAGCAG-3′ and 5′-CCAATAGGGCGAGGAGTGTG-3′. Taqman MGB probe sequences were lamin A (exon 11), 5′-ACTCGCAGCTACCG-3′; lamin C (exon 10), 5′-ATGCGCAAGCTGGTG-3′ and progerin (exon 11), 5′-CGCTGAGTACAACCT-3′. Reporter and quencher dyes for the LMNA locus assays were 5′ 6FAM and 3′- non-fluorescent quencher dye (NFQ; Applied Biosystems). 18 s (Assay HS_99999901_s1) probes and primers were provided by life technologies.