When I ran qPCR for chlorophyll-related genes from herbicide(Oxifluorefen) damaged rice plant, some genes showed multi Tm in the melting curve.
The result showed a more corrupted melting curve if they got severe damage.
But that same primer worked well with my co-worker's plant(hydroponic condition, not treated herbicide but lack of nutrients). It showed only one Tm in the result.
Has anyone had a similar experience?
Here I post some of my results.
If the primer was wrong(OsHO1), results would show like similar curve(also, in that graph, the more damaged samples had a high value of Tm2)
However, the results like CATc and PORB showed really messy curves, especially in OF treated sample(PORB Tm OF)
Now I'm arguing with chif senior. She blamed me it caused by my skill and asked me to find some research papers about this, but there were the same results even my senior did, and nobody wrote their fail in the report :(
Oh god of graduated students, please save my life.
Melt curves are the LEAST informative part of a qPCR experiment. No, they do not indicate anything about "DNA damage".
Multiple/messy peaks indicate that your amplicons are melting in more than one phase. Either the amplicons are too large (>150bp) or they have regions of very different %GC or both.
What do your Ct values look like? Your controls? Your primer efficiencies?
My advice is to read about how to properly set and and analyze a qPCR experiment. Don't make up reasons for messy data (DNA damage!), when the obvious answer is poor experimental design. Just because someone else used these primers/samples/etc. doesn't mean they did it correctly either.
Good luck.
Can you reproduce your colleagues ("good") results, setup a reaction like he/she did with their sample DNA and see if you can get similar results. It would also help to see a gel/capillary result for the amplicons. I am suspicious of the specificity of the primers, possible off-target amplifications? Are you using the same consumables, annealing/cycling protocols as your colleagues? PCR can be a very tricky thing, even a small difference such a difference the PCR mix used can make a difference I found.
Ok, looks like the real challenge here is getting quality data. Here's the thing, even if your target genes are impacted by the herbicide treatment, that difference will show up at a change in expression level, not in what the melt curves look like. Melt curves being messy means something needs optimizing, such as the annealing temp, amount of primer or template, length of amplicon etc. Did the previous person use the exact same primers you did? If so (and they left really good notes), try their protocol. I agree with Can Kiessling that PCR is a bit tricky, and small differences in the protocol can lead to big differences in the outcome.
Quick question: did your co-worker use the same rice cultivar that you are using? There could be sequence differences.
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