I don't know how frequent everyone is on this forum but I think this is the last straw for me regarding DNA methylation studies. I'm facing immense amount of problems in this area. I'm studying a gene DMBT1 and it's methylation status in saliva on volunteer samples. I'm not foraging into disease samples yet. Since I have no one to consult, my guide being new to epigenetics as well, I thought I'd post my problem here.
1. Found that I could design bisulfite specific primers and I used MethPrimer for it. Using the promoter sequence of DMBT1 I designed one set of primers.
Is it necessary to design more primer sets just to be on the safe side? My primers are
Left primer TGAATTTTTTAAGATTAAAATTAAGTT
Right primer AAAAAATAAAAACTTTTACTAACAATC
2. I used a Zymo EZDNA Methylation Direct kit which states that samples can be used directly for this kit and there's no need to extract DNA first. So I used DNA first. I used some previous good quality DNA too for conversion. But nothing worked in this kit and I have negligible purity of converted DNA. I ran the samples on a gel too and found nothing. Tried chilling the gel in EtBr buffer also and again 0 results. The 260/280 falls between 0.5 to 0.9 with some of the samples' ratio falling between 1.1 to 1.5. I have used freshly prepared CT conversion reagent yet I'm facing the same problems. The concentration of the samples is between 20-70ng/ul
3. Now, I used some of the samples to run a bisulfite PCR (only the samples with the highest purity i.e. ~1.5 260/280) and again 0 bands. I tried a gradient, again 0 bands, changed a number of cycles from 35-40 cycles. I'm using EpiMark Hot Start taq pol for the reactions since many papers have mentioned using a hotstart taq pol. In the papers I've read, they mention 72 degrees as the extension temperature, but in the booklet of the HotStart taq they mention using 68 degrees. i'm using 72, could this be a problem? I have tried a positive control using GAPDH and unconverted sample but all the reagents were same, I got bands. Now, I'm using two different PCR cyclers for my reactions.
4. I'm now starting with touchdown PCR the first reaction of which, didn't work. I've put another one on a different temperature gradient. I'm not immensely hopeful of the results of this one either.
Can anyone suggest what could be going wrong? Also, what else can I try regarding the PCR because my time is running out. i have only a couple of months to finish complete sequencing of 30 samples.