Quantification of Cellular Senescence Markers

ASCs were collected and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA samples were pre-treated with deoxyribonuclease I (Invitrogen Life Technologies, Carlsbad, CA, USA), and a SuperScript kit (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to synthesize cDNA according to the manufacturer’s recommendations. qRT-PCR was analyzed using miScript SYBR Green PCR Kits (Qiagen). Levels of cell cycle markers (p16, p21, and p53) mRNAs were determined by ABI PRISM 7700 cycler (Applied Biosystems, Foster City, CA). Each sample was analysed in duplicate with ribosomal 18S RNA as an internal control. Using telomeric primers, primers for the reference control gene (mouse 36B4 single copy gene), qPCR settings were performed as previously described [12 (link)]. The relative telomere length was shown as a T/S ratio indicative from the single copy gene. All fold changes in gene expression were determined using the 2 − ΔΔCT method. The values are presented as the mean ± SEM. All primers are listed in Supplemental Table 1.