The combination of several markers would be better. If looking only at mRNA level I suggest using p21 and DCR2 in addition to p16. You an also use SASP related genes, like IL-6 and IL-8.

Many thanks both :)

Since the activity of beta-galactosidase is a very useful mark of cellular senescence, and considering that you are looking for a mRNA mark, I would consider quantifying the mRNA for this enzyme.

Actually p16 may not be much good at the RNA level. p16 mRNA seems to rise before p16 protein and before cells actually senesce. p21 likewise, not much RNA change at the point of senescence. The changes are bigger at the protein level.

If you really want an mRNA marker, you may have to study what changes in your specific cell type. However some SASP products like IL6 and IL8 do change transcriptionally quite a lot, in the cells we have seen - as Valery K mentioned too. Jerry Shay has reported ISG15 mRNA as a marker of short telomeres.

Is that ISG15 mRNA present in mouse cells as well, since somatic cells in mice do have an active telomerase?

Alterations in gene expression vary with cell types and the signaling pathways that induce senescence-associated cell cycle arrest. Therefore, if you are specific about cell type(s) and signaling pathway(s), it may be easier to respond to your question. In general, as stated above by others, changes in RNA levels are associated with SASP and not with cellular or replicative senescence.

Alberto - don't know for sure, but I think only human cells were studied. One would expect that where cells senesce without short telomeres (as mouse cells often do), then they would be unlikely to express a marker of short telomeres.

Try to look the expression of transposons. It seems that their enhanced activity (and thus the genome mutability) is characteristic for senescence.

Please, see the article: Genomes of replicatively senescent cells undergo global epigenetic changes leading to gene silencing and activation of transposable elements.

De Cecco...[ J. Sedivy] Aging Cell 2013

Thanks all, much food for thought.

Hallo

We performed a study of senescence with p21(WAF1/CIP1) cyclin-dependent kinase inhibitor (CDKI) gene which has been considered to be a senescence marker.

Increased expression of the gene has been detected in vitro with cell replicative senescence experiments and in vivo among animals of advanced age. See atached paper. Also staining with beta Gal could give you some hints. All the best, Michaela

Also staining with beta Gal could give you some hints. Dear Michaela,

what you mean by some hints? I think that this is a marker rather for autophagy and may be positive in not quite terminally arrested cells.

Dear Jekaterina

The beta galactosidase staining stains the senescent cells in blue. A quite old paper describes the method:

Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O (September 1995). "A biomarker that identifies senescent human cells in culture and in aging skin in vivo". Proc. Natl. Acad. Sci. U.S.A. 92 (20): 9363–7. doi:10.1073/pnas.92.20.9363. PMC 40985. PMID 7568133..

The phenomenon is explained by the overexpression and accumulation of the endogenous lysosomal beta-galactosidase specifically in senescent cells.

Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (April 2006). "Senescence-associated beta-galactosidase is lysosomal beta-galactosidase". Aging Cell 5 (2): 187–95.

Years ago, a study performed in Tel Aviv has shown the correlation of beta Gal with telomeres shortening and gene expression in kidney cells.

Warmest regards, Michaela

Dear Jekaterina

The protocol for staining with beta Gal for detecting cell senescence could be found at the following link:

http://www.bio-protocol.org/wenzhang.aspx?id=247#.U5AaPKygbsQ

Warmest regards, Michaela

Thanks for advice all. We only currently have snap frozen tissue samples so beta-gal staining isn't appropriate, hence asking about RNA markers.

Dear James,

all cdk inhibitors, cytokines IL1, IL6, IL8, CSF2, GDF15, targets of TNFalpha. STAT and TGFbeta signaling pathways are potentially useful RNA marker candidates - based on in vitro studies, of course not strictly senescence-specific. You can also look to microarray studies - e.g. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD. Microarray analysis of replicative senescence. Curr Biol 1999; 9(17): 939-45 and later ones.

Zdenek

Many thanks

Hi James,

we did not confirm upregulation of p16 in senescent cells. Some histone clusters seem to be much promising markers (HIST1H1C etc).

Alena

Dear Michaela,

beta gal staining works better on fresh samples. As your samples are snap frozen it may be hard to do it. As you said p16 expression data should help. If you have enough sample, westerns for phospho p53 and p21might further help or look for Senescence associated heterochormatin foci! Cell cycle analysis along with above mentioned Westerns and gene expression should make your point that most of your cells are senescent!

Best wishes,

Kodanda