3 replies to this topic

[nisot]

My supervisor and I are both new to methylation analyses and we are planning on carrying out methylation analysis on DNA from a pure population of particular somatic stem cells. Problem is these cells are like 1% of the total cells in the organ so the amount of DNA would be quite minute. we are thinking of doing whole genome methylation analysis using MeDIP or Bisulphite. preferably MeDIP but we have no idea how feasible this is or other WGA options we have if any. Please suggestions and clarifications would be appreciated  

[et2b]

nisot, on Nov 7 2009, 12:06 AM, said:

My supervisor and I are both new to methylation analyses and we are planning on carrying out methylation analysis on DNA from a pure population of particular somatic stem cells. Problem is these cells are like 1% of the total cells in the organ so the amount of DNA would be quite minute. we are thinking of doing whole genome methylation analysis using MeDIP or Bisulphite. preferably MeDIP but we have no idea how feasible this is or other WGA options we have if any. Please suggestions and clarifications would be appreciated  

Hi,

Using the epitect 5hour FFPE protocol (so adding 100% ethanol extra) allows to bisulfite treatment and recover of minute amounts of DNA. We performed it successful for 50ng of gDNA, without carrier DNA. Below that you can use Ecoli gDNA as carrier.
Dont think you can do MeDIP on such small amount of DNA, but RRBS uses very little DNA. Maybe better alternative?
Best

[nisot]

et2b, on Nov 9 2009, 11:23 PM, said:

nisot, on Nov 7 2009, 12:06 AM, said:

My supervisor and I are both new to methylation analyses and we are planning on carrying out methylation analysis on DNA from a pure population of particular somatic stem cells. Problem is these cells are like 1% of the total cells in the organ so the amount of DNA would be quite minute. we are thinking of doing whole genome methylation analysis using MeDIP or Bisulphite. preferably MeDIP but we have no idea how feasible this is or other WGA options we have if any. Please suggestions and clarifications would be appreciated  

Hi,

Using the epitect 5hour FFPE protocol (so adding 100% ethanol extra) allows to bisulfite treatment and recover of minute amounts of DNA. We performed it successful for 50ng of gDNA, without carrier DNA. Below that you can use Ecoli gDNA as carrier.
Dont think you can do MeDIP on such small amount of DNA, but RRBS uses very little DNA. Maybe better alternative?
Best

Thanks for your answer. So after BS treatment, do you then whole genome amplify for downstream amplifications? such as whole genome seq?

[et2b]

nisot, on Nov 13 2009, 10:09 AM, said:

et2b, on Nov 9 2009, 11:23 PM, said:

nisot, on Nov 7 2009, 12:06 AM, said:

My supervisor and I are both new to methylation analyses and we are planning on carrying out methylation analysis on DNA from a pure population of particular somatic stem cells. Problem is these cells are like 1% of the total cells in the organ so the amount of DNA would be quite minute. we are thinking of doing whole genome methylation analysis using MeDIP or Bisulphite. preferably MeDIP but we have no idea how feasible this is or other WGA options we have if any. Please suggestions and clarifications would be appreciated  

Hi,

Using the epitect 5hour FFPE protocol (so adding 100% ethanol extra) allows to bisulfite treatment and recover of minute amounts of DNA. We performed it successful for 50ng of gDNA, without carrier DNA. Below that you can use Ecoli gDNA as carrier.
Dont think you can do MeDIP on such small amount of DNA, but RRBS uses very little DNA. Maybe better alternative?
Best

Thanks for your answer. So after BS treatment, do you then whole genome amplify for downstream amplifications? such as whole genome seq?

Meissner et al. Nature Methods, very very recently, everything you need.
bye bye, good luck and more of this kind of encouragements