EZ DNA methyaltion kit...trouble shooting - (Apr/21/2009 )
Dear All,
I need some help working with EZ DNA methylation kit. I wanted to bisulfite convert the FFPE material to study methyaltion status further.
So i have tried the EZ methylation start up kit, which also provided with the universal methylated genomic DNA and control primers hMLH1, hMLH2 to check for the convertion whether it worked or not.
After the bisulfite convertion i have done the control PCR reaction with the above mentioned primers (which should yield a product of 182 bp..if the convertion is complete), but i could only see the product formation in the positve control but not with my FFPE material ??? I have checked the quality of the FFPE DNA using oligo's pairs not related to a mehylated related regions that yileds a 160 bp product and the amplification worked on FFPE material and i could see the product.
Its really an essential step to confirm the bisulfite convertion to proceed further to avoid false positive results. Can someone help me how can i confirm the bisulfite convertion ??? is there any thing which i can change in my PCR conditions etc...i have used 2 µl of bisulfite converted DNA for the control PCR reaction and the reaction as follows, 95 for 10 min, 94.5 for 30 sec, 59 for 30 sec, 72 for 60 sec, 72 for 7 min, 35 to 40 cycles...4 degree hold..
FOr the bisulfite convertion...as mentioned in the protocol i used...98 for 8 min, 64 for 3.5 hours, 4 degree hold.
The positive control worked fine??? (this confirms the bisulfite convertion) y not in FFPE DNA ??? ..I have used the unconverted DNA as negative control and ofcourse i could not see any product with that ...Your suggestions are appreciated...Thanks and regards, Ra
When you check the quality of your ffpe DNA. How fragmented is it?
Your control assays should work give the size.
The fact that your control worked would suggest a sample type issue.
If your ffpe samples are highly fragmented. There is less chance of having the template there to amplify from.
Nick
RKA on Apr 21 2009, 12:59 AM said:
I need some help working with EZ DNA methylation kit. I wanted to bisulfite convert the FFPE material to study methyaltion status further.
So i have tried the EZ methylation start up kit, which also provided with the universal methylated genomic DNA and control primers hMLH1, hMLH2 to check for the convertion whether it worked or not.
After the bisulfite convertion i have done the control PCR reaction with the above mentioned primers (which should yield a product of 182 bp..if the convertion is complete), but i could only see the product formation in the positve control but not with my FFPE material ??? I have checked the quality of the FFPE DNA using oligo's pairs not related to a mehylated related regions that yileds a 160 bp product and the amplification worked on FFPE material and i could see the product.
Its really an essential step to confirm the bisulfite convertion to proceed further to avoid false positive results. Can someone help me how can i confirm the bisulfite convertion ??? is there any thing which i can change in my PCR conditions etc...i have used 2 µl of bisulfite converted DNA for the control PCR reaction and the reaction as follows, 95 for 10 min, 94.5 for 30 sec, 59 for 30 sec, 72 for 60 sec, 72 for 7 min, 35 to 40 cycles...4 degree hold..
FOr the bisulfite convertion...as mentioned in the protocol i used...98 for 8 min, 64 for 3.5 hours, 4 degree hold.
The positive control worked fine??? (this confirms the bisulfite convertion) y not in FFPE DNA ??? ..I have used the unconverted DNA as negative control and ofcourse i could not see any product with that ...Your suggestions are appreciated...Thanks and regards, Ra
RKA on Apr 21 2009, 10:59 AM said:
I need some help working with EZ DNA methylation kit. I wanted to bisulfite convert the FFPE material to study methyaltion status further.
So i have tried the EZ methylation start up kit, which also provided with the universal methylated genomic DNA and control primers hMLH1, hMLH2 to check for the convertion whether it worked or not.
After the bisulfite convertion i have done the control PCR reaction with the above mentioned primers (which should yield a product of 182 bp..if the convertion is complete), but i could only see the product formation in the positve control but not with my FFPE material ??? I have checked the quality of the FFPE DNA using oligo's pairs not related to a mehylated related regions that yileds a 160 bp product and the amplification worked on FFPE material and i could see the product.
Its really an essential step to confirm the bisulfite convertion to proceed further to avoid false positive results. Can someone help me how can i confirm the bisulfite convertion ??? is there any thing which i can change in my PCR conditions etc...i have used 2 µl of bisulfite converted DNA for the control PCR reaction and the reaction as follows, 95 for 10 min, 94.5 for 30 sec, 59 for 30 sec, 72 for 60 sec, 72 for 7 min, 35 to 40 cycles...4 degree hold..
FOr the bisulfite convertion...as mentioned in the protocol i used...98 for 8 min, 64 for 3.5 hours, 4 degree hold.
The positive control worked fine??? (this confirms the bisulfite convertion) y not in FFPE DNA ??? ..I have used the unconverted DNA as negative control and ofcourse i could not see any product with that ...Your suggestions are appreciated...Thanks and regards, Ra
Hi Nick,
I dont know how fragmented is my FFPE DNA..i have just checked the quality by using other set of primers which yields a 160 bp product and which worked..whts ur suggestion to check the fragmentation ??? or degradation ??? thanks
running your FFPE sample on a gel would help. Depending on how much you have obtained.
I suspect your samples have been chewed up by the conversion process. but as pcrman says, you need to give us more information.
methylnick on Apr 22 2009, 09:13 AM said:
I suspect your samples have been chewed up by the conversion process. but as pcrman says, you need to give us more information.
i dont have much FFPE dnas to check on the gel !!!!!