I am attempting to do bisulfite sequencing on a frozen lung section extracted by laser capture microdissection. So far, I have not had good results getting past the PCR amplification step. To isolate DNA from LCM, I am using the qiagen micro kit. I've been getting between 1-15 ng total DNA from these extractions. This is normal because I am only taking between 40-200 cells from LCM, and I cannot increase the amount of tissue I extract from LCM. Then I am using the Qiagen bisulfite kit to do the CT conversion with a DNA protect buffer so I can keep the fragments intact. Following this, I am amplifying the converted DNA with the Whole Bisulfitome kit from Qiagen. This tissue is what I use for the PCR of our region of interest, and I have not been getting any results. The primers are known to work on bisulfite converted DNA, and I have successfully run it on control DNA. Does the fact that the DNA strands after whole genome amplification are double stranded instead of ssDNA have anything to do with this? Normally, the bisulfite DNA would be single stranded by the time it goes through the PCR. The PCR stages include a denaturation step so I couldn't see why it would be a problem...
Hi John,
Because you're starting with very low amounts of DNA, I recommend checking DNA concentration and quality after both bisulfite treatment and whole genome amplification to make sure you have an adequate amount of template for the PCR.
You also might need to increase the number of PCR cycles or perform a nested PCR reaction for higher product amplification. You can design a nested PCR to first amplify your gene of interest with methylation-insensitive primers between a region spanning the target methylation site. The product should be around 200 bp and gel-extracted to improve RT-PCR efficiency. Then you can perform a second reaction on your first-step product with methylation-specific primers.
Since you are working with low input DNA, it is critical to design a small PCR amplicon (must be smaller than 150 bp, ideally smaller than 100 bp). Before analyzing LCM samples, it is recommend to validate the PCR primer sets to avoid the preferential PCR amplification with low input DNA.
Liying Yan The primers I am using are 500 bp. If you know any primer sets that have been used before on human tissue which are smaller i'd be interested in trying new primers!
Hi John,
Are you working a specific gene? 500 bp for bisulfite PCR is too big for regular input DNA. Thanks.
Liying Yan The gene is SMAD3, and has been successfully performed through sequencing by one of our collaborators, but not on LCM extracted DNA. However, I am performing a feasibility study and don't need to use SMAD3 specifically.
For a feasibility study, Line-1 Pyrosequencing methylation analysis may be a good start. The Line-1 Pyrosequencing assay can detect less than 1 ng input DNA.
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