3 replies to this topic

[GNANA]

Hi everybody..,
To be honest am brand new to this technique...i got to screen methylation status for the promoter of my gene of interest.  i got primers thru methprimer design tool, and got the quiagen epitect bisulphite conversion kit and quiagen MSP kit, now am half the way, i followed the quigen protocol for bisulphite conversion and now need help to proceed the bisulphite converted DNA for MSP.  my questions are,
1) How much starting template (bisulphite converted) i have to use for MSP(i used 200 ng for Bisulphite conversion),
2) what all controls i have to use.
3) how to interpret my result (am expecting Hypomethylation in my gene promoter).
4) Is MSP is enough to screen or shd i hav to do sequencing also to have a convincing Data.

thanks in advance for your valuable suggestions.

Gnana...

Edited by GNANA, 16 September 2010 - 06:00 AM.

[methylnick]

Welcome to Bioforym GNANA!

GNANA, on 16 September 2010 - 05:21 AM, said:

1) How much starting template (bisulphite converted) i have to use for MSP(i used 200 ng for Bisulphite conversion),

usually 10-20ng per PCR is suffice for MSP

GNANA, on 16 September 2010 - 05:21 AM, said:

2) what all controls i have to use.

known methylated and unmethylated samples, a good place to start is the Qiagen Epitect DNA Methylation controls, they are human, if you are working in non-human, then artificially methylated (with SssI) and unmethylated (whole genome amplified DNA) DNA.

GNANA, on 16 September 2010 - 05:21 AM, said:

3) how to interpret my result (am expecting Hypomethylation in my gene promoter).

PCR amplicon with your unmethylated primer set and none in your methylated primer set.

GNANA, on 16 September 2010 - 05:21 AM, said:

4) Is MSP is enough to screen or shd i hav to do sequencing also to have a convincing Data.

That really depends on what you want to show, MSP can be fiddly, in that you are likely to get amplicons with both primers, and you will need to tweak the PCR conditions so you get the result you are after. With MSP you are only testing the methylation status of CpG's hybridised by the primer's themselves and you are inferring methylation of CpGs within the amplicon. Sequencing will alleviate this.

Good luck!

[GNANA]

thanks a lot methylnick....i jus finished my MSP., got no bands in any lane...i explain you how i did...
i selected a normal cell line tat express basal level of my gene of interest as a comparison control for my gene tat express more in cancer cell lines. so first inorder to optimize MS-PCR i used three points, one is the DNA of normal cell line bisulphite converted (200 ng- qiagen epitect Bis conv. kit- eluted 20 micro litres final, from which i used 6 micro litres for PCR),second is the non converted same cell line (used 25ng Dna for PCR), third is unmethylated control supplied by qiagen (10 ng -suggested by qiagen).

The only change i did was instead of 50 Micro litres, i used 25 final volume for PCR, i dnt know whether this could be the reason, bcoz on seeing the gel it sounds like PCR failure, as there is no band in the Unmeth control itself.  

PCR Reaction: (qiagen MSP kit)
Template : 6 micro litres  (from 200 ng bis converted and eluted 20 micro final, i dnt know is it possible to quantify the bis convted Dna, so )
Prim U/M F : 0.4 micro molar final conc.
Prim U/M R : 0.4
Msp Master Mix(2x): 12.5 micro litres
Water : to the final 25

Thermal protocol:

95-10 mins

94-15 sec
51-30 sec
72-30 sec
x 35 cycles

72-10 mins

Amplicon size 110 bp.

Kindly suggest me to optimize the protocol for MSP. also enlighten me which all points to use; i mean samples i can use for PCR optimizaton alone, may be is the meth or unmeth control alone is enough for optimization or do i hav to run my bis conv Dna also along?.
let me kno if i need to provide any other details..
thanks a lot for any of ur suggestions...

is it right to amplify the promoter region of my gene of interest and then continue with the bisulphite conversion and MSP or hav to do with the whole genome only???

Gnana...,

Edited by GNANA, 17 September 2010 - 11:01 AM.

[GNANA]

sorry, for the above the amplicon size is 281 bp not 110 as i mentioned above....

Edited by GNANA, 19 September 2010 - 02:39 AM.