If I check the amplicon through MFold from IDT website and find that the Tm of the amplicons secondary structure is around 40C, will it affect the mobility of the products in agarose gel electrophoresis? The amplicons size should be 137bp but the gel image showed ~210bp and there is another band at the bottom ~ 100bp.
Please refer to the figure below (Lane 2 and 3 from the right).
Well, Shin, DNA in a standard TAE agarose gel runs as double-stranded fragments, since you don't do anything to denature it, with little possibility to make additional secondary structures, mostly when they are so short (longer pieces can bundle and intertwine, so it is recommended to run them with low current to better focus the band).
My interpretation (with the little information provided...) would be that your PCR is highly unspecific and I would check the primers (you can also try single primer PCR, often primers can work unspecifically as sense and antisense... it's a good indication that they should be re-designed!). With this starting point it does not look like any PCR protocol improvement would help much (annealing temperature increase, annealing time elongation, step-down approach, etc)...
Yes, definitely. Check out this very good publication on the topic: Viovy et al, Electrophoresis, 14, 1993, 322.
Basically, all fragments will migrate with an apparent size. When you place yourslef in denaturing conditions (e.g. urea or guanidinium hydrochloride), you linearize most of the fragments, but this is not 100 % efficient.
From what you describe, though in your gel. You may have dimerization and degradation, though. The sizes are very different from the expected value...
Well, Shin, DNA in a standard TAE agarose gel runs as double-stranded fragments, since you don't do anything to denature it, with little possibility to make additional secondary structures, mostly when they are so short (longer pieces can bundle and intertwine, so it is recommended to run them with low current to better focus the band).
My interpretation (with the little information provided...) would be that your PCR is highly unspecific and I would check the primers (you can also try single primer PCR, often primers can work unspecifically as sense and antisense... it's a good indication that they should be re-designed!). With this starting point it does not look like any PCR protocol improvement would help much (annealing temperature increase, annealing time elongation, step-down approach, etc)...
Hi, Piertro.
Thank you very much for the advice. May I know how to check primers' quality? My primers are as below:
Forward: 5'-tcattttgacggaaatggcaacc-3'
Reverse: 5'-aaggaacatagcttcaaccgcag-3'
But I have checked the primers with positive control (HepG2). It actually showed 137bp band size with the same condition with my sample (Macrophage) . May I know what is the problem?
Ok Shin, I think I understand that your primers give a specific band using a control cDNA. The primers by eye look pretty much ok, maybe a bit too strong at their 3' end, which can induce unspecific annealing and amplification in certain conditions.
Anyway, if in your positive control they were specific (one single band of 130-140 bp), then you should doubt of your macrophage RNA extraction. Load 0.5-1 ug of the RNA on a 1.5% TAE agarose gel (in normal DNA loading buffer) and check its quality by looking at the relative intensity of the 28 and 18S rRNA bands (first should be double intensity of second) and the presence and intensity of any smear (should be very weak, mRNA is 2-5% of rRNA). If these two parameters are not as expected, then you simply have a degraded RNA to start with, which means you'll probably have poor cDNA and no specific template, therefore your primers will take advantage of unspecific binding.
Keep in mind that also the chemical quality of your RNA is important, even if it is not degraded. Depending on the preparation method, you might carry on different types of contaminations (guanidine isotiocyanate, phenol, resins). If you have a Nanodrop or similar spectrophotometer make a scan analysis and look at the Absorbance ratios 260/280 (should be 2-2.1) and 260/230 (should be above 2.2). If they are lower you might have protein/phenol contamination in the first case and guanidine in the second... Phenol you can remove by CHCl3 extractions and re-precipitation in EtOH, guanidine you can only remove by doing a LiCl precipitation of your RNA, which is done with 2.5-5 M LiCl in water (prepare a 2x LiCl stock solution, and add equal volume to your RNA) at -20 C for 30-60 min. and spinning 30 min at 4 C.
I hope it helps... good luck.
I agree with Pietro, it really looks like you have PCR issues not migration.
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