This will be long, but I am out of ideas and require help. I am having difficulty with my real time PCR as I am obtaining multiple products in each sample. I am looking for expression of JDP2 in response to radiation in human cells. I initially tested several primers, and chose the one which resulted in one consistent product across all samples. The amplicon length of this primer is 136. Using Beta actin as a control, I did a real time PCR using the samples.
~Timeline of Experiment~
*Isolated RNA using trizol.
*Reverse transcription according to the recommended Superscript II protocol from ThermoFisher. Used 200ng RNA.
*Used equivalent of 5ng per well of “cDNA” for the real time PCR, using recommended SYBR green protocol from Applied Biosystems.
*Most samples are fine and do not have multiple products, some others do have multiple products. However, what is most concerning to me is that the same product that is consistent in all sample also appears in my non-template control wells. I am using a new plate, pipette tips, master mix, and primer. The only thing that is consistent is the nuclease free water I’m using.
The attached Stepone .eds files are
1. The initial primer test.
2. Expression Test
3. Expression Test
4. Expression test (using more RNA per well)
5. Temperature gradient at 50, 54, 58, 62, 66, 70 Degrees C
What could be my problem? If you have any ideas please share, and if you have any questions, please ask. I will respond promptly.
Thank you so much for any help.
Having trouble viewing the .eds files, but from what you've stated it sounds like Primer Dimers is a good possibility to explain what you are seeing. I would run the final product after your RT-PCR out on an agarose gel in order to confirm what you say is the consistent product is actually 136bp as you expect and not smaller, which would suggest primer dimers; especially since you are seeing the same product in the no-template control.
Dear Jacob.
- I cannot open your eds files from my current viewing platform
- However I shall posit done general points:
- If bands appear in all samples as well as your NTC that suggests primer dimer/oligomer issues with those particular primers
- The best solution to this problem is to redesign primers or use alternative primers
- bands in NTC samples suggesting the presence of primer dimers can be screened out with freeware such IDT oligo design tool, viz
Dear Jacob,
I think you have a problem with primers design or cDNA template.
1. Firstly try to check your primers for dimers or other secondary structure. You can use the Oligoanalizer online tool or other i.e. NetPrimer. Check the harpins , selfdimers and heterodimers structurs , generaly look on deltaG value, which should be less negative than -6, but deltaG value up to -9 sometimes give also good results, hard to explain. If deltaG values are poor maybe good way is to re-design your primers.
2. If secondary structures are okey, think about localization of primers. It should be on exon-exon junction, If primers are situated in the middle of exons in the PCR reaction you can also amplified other amplikons i.e. pseudogenes. The next step you can do is to thinkig about your cDNA samples were contaminated by genomic DNA and primers amplified this template, so you can think about add DNAase treatment after RNA isolation.
Maybe useful will be done agarose gel electrophoresis to compare with DNA Ladder Standard the size of your product? Then you can go to Primerblast tool on NCBI web site, paste the primers sequence and check possible product (verify the specificity of the primers).
Good luck,
- Further to mine and Mareks comments the following site can be used to screen for primer dimers
- https://www.idtdna.com/calc/analyzer
- https://www.idtdna.com/pages/support/technical-vault/faq/application/how-can-i-check-my-pcr-primers-using-the-oligoanalyzer-program-to-ensure-there-are-no-significant-primer-design-issues-
- I find the following program more convenient for self dimers and hair pin loops
- http://biotools.nubic.northwestern.edu/OligoCalc.html
If it is a primer dimer then running of RT-PCR product in agarose gel electrophoresis and after that measuring of length using Alpha Ease FCTM software for authentic one would possibly be a solution.
The hetero and homo-dimer analysis leads me to believe that the product is not due to primer-dimer formation (attached). I will run the PCR products on agarose gel as suggested and hope to see a band at 136bp.
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