Universal Unmethylated Control - (Feb/09/2010 )

Hello, I recently bought CpGenome universal unmethylated DNA set from Millipore, to use as a control in MSPCR. The set includes one vial of human genomic DNA and another of human fetal cell line DNA. Are both of these samples suitable for use as an unmethylated control? Is one better than the other? If someone could help me with this, it would be greatly appreciated!

It really depends on your gene(s) of interest.

The fetal cell line DNA in theory is mostly hypomethylated but not so at imprinted genes for instance. Two samples are included so that most loci looked at by researchers are unmethylated in at least one of the samples in the kit.

Sucky I know

Thanks methylnick! I ask because I did bisulfite modification using Qiagen Epitect Kit and then MSPCR, but have continued to get confusing results.

For the methylated control, I am getting a strong band of the expected size using methylated-specific primers, and no band using unmethylated primers. For the human genomic AND fetal cell unmethylated DNA control, however, I see a strong band from the methylated-specific primers and either a weak or nonexistant band using unmethylated-specific primers.

The primers I am using for my 2 genes of interest are published. I made several modifications to the bisulfite kit protocol in hopes of getting more conversion and yield. These include:
-extending the modification cycling by 5min at 95deg and 2hr at 60deg
-during cleanup, adding 560ul Buffer BL and 560ul 100% EtoH, incubating at RT for 10min, then spinning down onto the column in two steps
-incubating elute on column for ~5min
-eluting with 40ul Buffer EB

PCR: For gene1: 10min at 94deg, (30sec at 94, 45sec at 63, 45sec at 72deg) X 35cycles, 5min at 72deg
For gene2: 10min at 94deg, (30sec at 94deg, 30sec at 56deg, 45s at 72deg) X 35cycles, 7min at 72deg

One thing I have not done, however, is use Hotstar Taq or 'Q solution' for the PCR. Do you think these would make a significant difference? Has anyone else used these controls and been successful? I appreciate the help!

I am also facing almost similar problem I am using Qiagen Epitect bisulfte kit and chemicon universal methylated and unmethylated controls. after bisulfite treatment and using my gene of interest primers I m getting very strong band with methylation primers in universal methylated,unmethylated controls and all other samples and no band with unmethylated primer set I am wrried with the strange results i should get band with unmethylaed primer and not with methylated primer in universal unmethylated control but I am getting reverse. I will be so greatful if anyone can suggest me some remedy.

Thanks

KD123 on Feb 11 2010, 04:18 PM said:

Thanks methylnick! I ask because I did bisulfite modification using Qiagen Epitect Kit and then MSPCR, but have continued to get confusing results.

For the methylated control, I am getting a strong band of the expected size using methylated-specific primers, and no band using unmethylated primers. For the human genomic AND fetal cell unmethylated DNA control, however, I see a strong band from the methylated-specific primers and either a weak or nonexistant band using unmethylated-specific primers.

The primers I am using for my 2 genes of interest are published. I made several modifications to the bisulfite kit protocol in hopes of getting more conversion and yield. These include:
-extending the modification cycling by 5min at 95deg and 2hr at 60deg
-during cleanup, adding 560ul Buffer BL and 560ul 100% EtoH, incubating at RT for 10min, then spinning down onto the column in two steps
-incubating elute on column for ~5min
-eluting with 40ul Buffer EB

PCR: For gene1: 10min at 94deg, (30sec at 94, 45sec at 63, 45sec at 72deg) X 35cycles, 5min at 72deg
For gene2: 10min at 94deg, (30sec at 94deg, 30sec at 56deg, 45s at 72deg) X 35cycles, 7min at 72deg

One thing I have not done, however, is use Hotstar Taq or 'Q solution' for the PCR. Do you think these would make a significant difference? Has anyone else used these controls and been successful? I appreciate the help!