Hi, I am testing a new technology to measure methylation level and I am looking for some control sample/DNA. The ideal one would be two cell lines (since they're easier to manipulate than tissue), one fully methlylated, and the other is fully unmethylated. Anybody has suggestions what cell line and what gene shall I use? Thanks.
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#2
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Posted 06 March 2009 - 05:42 AM
hhh, on Jan 14 2005, 10:44 PM, said:
Hi, I am testing a new technology to measure methylation level and I am looking for some control sample/DNA. The ideal one would be two cell lines (since they're easier to manipulate than tissue), one fully methlylated, and the other is fully unmethylated. Anybody has suggestions what cell line and what gene shall I use? Thanks.
Cell lines??? I would use whole genome amplification to generate completely unmethylated DNA and SssI enzyme to generate 100% methylated DNA. No growing of cells!
Than just mix to make 0%, 10% 20% etc etc mixture. Very nice way to determine quality of your technique. than use gene like Leptin etc etc of which a lot of in vivo methylation data is available, to make your abstract key words nice and sexy when publishing
Looking forward to your publication
#3
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Posted 06 March 2009 - 05:44 AM
et2b, on Mar 6 2009, 02:42 PM, said:
hhh, on Jan 14 2005, 10:44 PM, said:
Hi, I am testing a new technology to measure methylation level and I am looking for some control sample/DNA. The ideal one would be two cell lines (since they're easier to manipulate than tissue), one fully methlylated, and the other is fully unmethylated. Anybody has suggestions what cell line and what gene shall I use? Thanks.
Cell lines??? I would use whole genome amplification to generate completely unmethylated DNA and SssI enzyme to generate 100% methylated DNA. No growing of cells!
Than just mix to make 0%, 10% 20% etc etc mixture. Very nice way to determine quality of your technique. than use gene like Leptin etc etc of which a lot of in vivo methylation data is available, to make your abstract key words nice and sexy when publishing
Looking forward to your publication
silly me, this dates back to 2005!!! better to get back to writing my %^&# article (procrastination rules!!)
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Posted 07 March 2009 - 09:47 AM
et2b, on Mar 6 2009, 08:14 PM, said:
et2b, on Mar 6 2009, 02:42 PM, said:
hhh, on Jan 14 2005, 10:44 PM, said:
Hi, I am testing a new technology to measure methylation level and I am looking for some control sample/DNA. The ideal one would be two cell lines (since they're easier to manipulate than tissue), one fully methlylated, and the other is fully unmethylated. Anybody has suggestions what cell line and what gene shall I use? Thanks.
Cell lines??? I would use whole genome amplification to generate completely unmethylated DNA and SssI enzyme to generate 100% methylated DNA. No growing of cells!
Than just mix to make 0%, 10% 20% etc etc mixture. Very nice way to determine quality of your technique. than use gene like Leptin etc etc of which a lot of in vivo methylation data is available, to make your abstract key words nice and sexy when publishing
Looking forward to your publication
silly me, this dates back to 2005!!! better to get back to writing my %^&# article (procrastination rules!!)
#5
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Posted 07 March 2009 - 09:57 AM
Hi, I am trying to design my BSP experiment with the help of the mutual valuable informations mentioned in this forum.Yet no one here talk about what control is usually used in BSP.Hope to have a sufficient answer about this, as I am the only fellow doing BSP in my lab .
#6
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Posted 08 March 2009 - 11:47 PM
taurus2009, on Mar 7 2009, 06:57 PM, said:
Hi, I am trying to design my BSP experiment with the help of the mutual valuable informations mentioned in this forum.Yet no one here talk about what control is usually used in BSP.Hope to have a sufficient answer about this, as I am the only fellow doing BSP in my lab .
For bisulfite sequencing you could use a whole genome amplified sample. PCR products are 100% garantueed DNA methylation free. But, the only thing you really really really really need to check when doing BSP --> look in the sequencing track if the non CpG Cytosines are 100% converted by the bisulfite reaction. If not, you need to re-do the bisulfite treatment, any BSP quantifications of cytosines are then bullshit.
hope this helps
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Posted 10 March 2009 - 04:42 AM
et2b, on Mar 9 2009, 12:47 AM, said:
taurus2009, on Mar 7 2009, 06:57 PM, said:
Hi, I am trying to design my BSP experiment with the help of the mutual valuable informations mentioned in this forum.Yet no one here talk about what control is usually used in BSP.Hope to have a sufficient answer about this, as I am the only fellow doing BSP in my lab .
For bisulfite sequencing you could use a whole genome amplified sample. PCR products are 100% garantueed DNA methylation free. But, the only thing you really really really really need to check when doing BSP --> look in the sequencing track if the non CpG Cytosines are 100% converted by the bisulfite reaction. If not, you need to re-do the bisulfite treatment, any BSP quantifications of cytosines are then bullshit.
hope this helps
I dont get it: in parallel to your sample, you conduct Bisulfite treatment on DNA un-methylated and fully methylated.
At the end of the treatment I am planning to run several PCRs with the different bisulfite primers (for the region of interest I want to look at) and then send them for sequencing. Shall I run the same PCRs on my fully methylated and fully UNmethylated DNA?
Thanks for help.
J
