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DNA methylation analysis - doubts - (Aug/21/2008 )

Hi everyone,

I am a new PhD student in this field and i am doing some bioinformatic work before DNA methylation analysis. However, my current supervisor is not of this field either, and the one that will help me with this will only come in October. So, as you imagine, i have a lot of questions...
Could you help me?!

My first task was to search for CpG islands in several genes, and I used MethylPrimer Express. I am now trying to get MSP (or BSP, i am not sure yet which one i will use) primers. This software provides me already these primers but I am not sure if i can use them directly or if it is better to check them with another software. Do you recommend one?
I found 3: PerlPrimer, BiSearch and MSPprimer but I don't know exactly how they work because i can't find good tutorials.

Also, after designing the primers, which are the best companies to produce them?

Another question: is there any BSP/MSP technique that would allow me to analyze several genes at the same time? (I need to study methylation of 10 genes in 4 different tissues of CTRL versus diseased mice of 3 different ages and this would take me too long to study one by one!!!)

Thank you very much
Cheers
Cris

-EpiCris-

1. smart to optimize the conditions of MSP for each gene.
2. order the primers from IDTDNA, or others.
3. possible to test all at the same time. Recently I tested two genes at the same time for a pilot study. If you have any question, email me.
Good luck



QUOTE (EpiCris @ Aug 21 2008, 05:53 PM)
Hi everyone,

I am a new PhD student in this field and i am doing some bioinformatic work before DNA methylation analysis. However, my current supervisor is not of this field either, and the one that will help me with this will only come in October. So, as you imagine, i have a lot of questions...
Could you help me?!

My first task was to search for CpG islands in several genes, and I used MethylPrimer Express. I am now trying to get MSP (or BSP, i am not sure yet which one i will use) primers. This software provides me already these primers but I am not sure if i can use them directly or if it is better to check them with another software. Do you recommend one?
I found 3: PerlPrimer, BiSearch and MSPprimer but I don't know exactly how they work because i can't find good tutorials.

Also, after designing the primers, which are the best companies to produce them?

Another question: is there any BSP/MSP technique that would allow me to analyze several genes at the same time? (I need to study methylation of 10 genes in 4 different tissues of CTRL versus diseased mice of 3 different ages and this would take me too long to study one by one!!!)

Thank you very much
Cheers
Cris

-microlight-

you mean ,MSP is better ?




QUOTE (microlight @ Aug 23 2008, 12:03 AM)
1. smart to optimize the conditions of MSP for each gene.
2. order the primers from IDTDNA, or others.
3. possible to test all at the same time. Recently I tested two genes at the same time for a pilot study. If you have any question, email me.
Good luck



QUOTE (EpiCris @ Aug 21 2008, 05:53 PM)
Hi everyone,

I am a new PhD student in this field and i am doing some bioinformatic work before DNA methylation analysis. However, my current supervisor is not of this field either, and the one that will help me with this will only come in October. So, as you imagine, i have a lot of questions...
Could you help me?!

My first task was to search for CpG islands in several genes, and I used MethylPrimer Express. I am now trying to get MSP (or BSP, i am not sure yet which one i will use) primers. This software provides me already these primers but I am not sure if i can use them directly or if it is better to check them with another software. Do you recommend one?
I found 3: PerlPrimer, BiSearch and MSPprimer but I don't know exactly how they work because i can't find good tutorials.

Also, after designing the primers, which are the best companies to produce them?

Another question: is there any BSP/MSP technique that would allow me to analyze several genes at the same time? (I need to study methylation of 10 genes in 4 different tissues of CTRL versus diseased mice of 3 different ages and this would take me too long to study one by one!!!)

Thank you very much
Cheers
Cris


-zlx-0814-

Hi Cris,

I would recommend using BSP if you want to go for several CpGs in one Promoter. Both, MSP and BSP can be multiplexed, but you should be really really really optimise each gene's PCR. If you start with methylation analysis, go for one gene at a time. MSE is in my opinion very good for picking primers for methylation analysis.



Good luck, Kr├╝mel

-kr├╝melmonster-

Thank you both very much!

If the MPE is indeed a good tool to MSP/BSP design then i think i will first show to my Epigenetic supervisor the MPE primers and then producing them.
But anyway, I didn't understand what was SMART to check the primers...

About the analysis of the several genes, i was thinking to use MSP because it is less expensive and i am trying to introduce the technique in my lab. However, in addition to the need to do a lot of PCRs, i would have to quantify the results right? What do you suggest? to use a real time PCR?

Best regards
Sueli

-EpiCris-

I prefer qMSP (real-time PCR) to quantify what percentage of methylation rather than BSP (expensive), based on my experience.


QUOTE (EpiCris @ Aug 25 2008, 07:00 AM)
Thank you both very much!

If the MPE is indeed a good tool to MSP/BSP design then i think i will first show to my Epigenetic supervisor the MPE primers and then producing them.
But anyway, I didn't understand what was SMART to check the primers...

About the analysis of the several genes, i was thinking to use MSP because it is less expensive and i am trying to introduce the technique in my lab. However, in addition to the need to do a lot of PCRs, i would have to quantify the results right? What do you suggest? to use a real time PCR?

Best regards
Sueli

-microlight-