ABI have released MethylPrimer Express! - A new primer design program for DNA methylation....For Free!!! (Jun/02/2006 )

primers look good,

good luck with it!

Nick

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Hi,Nick

This week I started an initial amplification using BSP primers,then took 2~3ul PCR product as the template to perform the second amplification using MSP primers ,and I got the bands I wanted on the gel.Althought the bands I've gotten are not very bright under the UV light ,I think I can get better results throught adjusting the PCR conditions.

Tnank you very much for your help!


Best regards

rosylight

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Hi,I`m a newer.I have downloaded the software.It looks very good.But I don`t know how to use it to design the primer.I just want to find the CpG methylation in the promoter of the VHL gene.Who can help me?

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Hi,leydig

I've downloaded the directions of MPE.I can mail it to you if you want ,or you patse the sequence of your pet gene here ,I'm glad to have a try .

Good luck!

rosylight

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Hi all,

I have also some problems with my MSP. I hope, you can help me.

Does anybody know a program to design msp-primers and PROBES for taqman-PCR ?
I downloaded MethylExpress, but this program is only for sequencing the DNA-products, so it is impossible to generate probes.
I want to analyse the methylation status with a specific probe.

Our results in MSP are a bit funny....the "normal-PCR" runs great, but in the taqman-PCR, our samples don´t give good results. Only our calibrator shows good curves with the probe (so the probe works...but not authentic).

Does anybody know an other method to quantify the methylation status?

Looking forward to your reply.

Labgeg

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methylprimer express does do MSP primer pairs,

I am not aware of any software that picks a taqman probe for methylation.

you would probably have to do this yourself. just at a guess, design a taqman assay on your loci of interest with BEFORE bisulfite conversion and with the selected probes, mock bisulfte treat them and adjust the lengths of the probe to match the ideal conditions for a probe with all non CpG C's converted to T's and all CpGs either C for methylated or T's for unmethylated.

N

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[quote name='Labgeg' date='Aug 21 2006, 05:07 AM' post='64908']
Hi all,

I have also some problems with my MSP. I hope, you can help me.

Does anybody know a program to design msp-primers and PROBES for taqman-PCR ?
I downloaded MethylExpress, but this program is only for sequencing the DNA-products, so it is impossible to generate probes.
I want to analyse the methylation status with a specific probe.

:D I am also trying to use qPCR to quantify M vs U and I believe the syber green approach could be useful in tandem with MSP primers designed with Methprimers for qPCR of M/U CpG.

However I don't exactly how to express the results.

As anybody of you have a suggestion?

I believe that when you pick up the cells from you plate you have a balance between M and U DNA for a specific CG.
When you expect your gene to be upregulated due to a specific stimuli this should result in a shift from the predominant M vs a more U envirorment surrounding the promoter of your gene. This is what I observe but how could you quantitate this difference?

thank you for your help

Danilo

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rosylight and all
Thank you for your kindness.My Email:xuanhq@163.com.I will try my best.However I may meet with some problems.I`d like you all to help me.THanks again.

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Just used MPE to make some MSP primers. The reaction works well, just need to really work out the reaction conditions, especially primer concentrations in relationship to the annealing temp.

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Hi, rosylight,

I saw you got your bands by MSP. Now I have some problems with my MSP. Could you tell me how you get your genomic DNA and how you make the genomic DNA modification by bisulfite? How much genomic DNA you use for bulsifite modification?

Looking forward to hearing from you.

Thanks for your help

Pink

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