A question about sample preservation for BSP - (Aug/23/2007 )
Hi, everyone. I've been doing BSP for a year and it all went well thanks to the information on this forum.
Recently I worked on two samples of 1 year's preservation under -20 degree C and the results turned out to be a little surprising.
In short, I got less methylation CG than what I've anticipated. I can't figure out why but list some possibilities.
1. My anticipation was totally wrong, the results revealed the true methylation status of the sample.
2. Over convertion occured.
3. There was methylation loss during the preservation.
About 2, I used the ordinary 5M bisulfite/50 degree C/6h protocol and other parallel experiments didn't show any sign of over convertion. According to two early posts on this forum(post1 post2) it would need overnight or several days of reaction to over converted the sample, so it seems unlikely to be the cause of my problem.
What I am wondering is whether there is any possibility that samples under preservation could get methylaiton loss. Has anyone here been confronted with this problem? Any discussion would be much help to me. Thanks.
Hi Pan Lei,
we have seen something similar with old samples. I think, it would be better to store at -80°C to prevent spontaneous demethylation. Over conversion is not likely in my eyes.
K.
we have seen something similar with old samples. I think, it would be better to store at -80°C to prevent spontaneous demethylation. Over conversion is not likely in my eyes.
K.
Thanks a lot, krümelmonster. I will do more experiments to check this possibility although at present I have no idea why this demethylation could happen, scince rough treatment like boiling for 15 mins didn't give rise to this change.