4 replies to this topic

[cells]

Hi,

I am new to DNA methylation studies and I would appreciate a little help on this.  I'm just trying to standardize my techniques for RT-PCR and at the moment, I am looking at gene expression of a known methylated gene (GSTP1) in prostate cells (LNCaP).

What I'm finding a bit strange is that I am seeing expression of the control (untreated) cells in RT-PCR and Western analysis.  Could anyone suggest a possible reason for this.  There are papers that suggest GSTP1 should be silenced in this cell line.  I have also done treatments with 5aza-2'deoxycytidine and see very little change from the control.

Thanks!

[methylnick]

There is an underlying issue with cell lines with regard to contamination, is the cell line you think is LnCAP really LNCaP? The same could potentially be said with the papers that publish the finding you describe, were they really using LNCaP?

Nick

[ForEpi]

From personal experience I can testify that the GSTP1 promoter is nearly completely methylated (average of 92% over 14 CpGs) and is not expressed in LNCaP cells (it is in RWPE-1, PC3 and DU145 though). Expression can be induced by dAZA treatment.

Hope this helps.

Bram

[cells]

Thanks for the replies.

I do see a slight increase in GSTP1 and RASSF1 expression after 5azaDcd treatment but I still see some expression in the controls.  Is this normal?  Is there expected to be some expression at the basal level?

Also, is there any type of marker to make sure this is LNCaP cells?  I received these cells from someone else so there could be a mistake in its identity.

[ForEpi]

There is NO expression of GSTP1 in LNCaP cells and you should see indeed a slight increase using dAZA. My cells came straight from ATCC and were mycoplasma checked every month. You might want to check them using an STR forensic kit.

Bram