Hi, I wonder if it is possible to detect the presence of certain cytokines (such as INF-y or IL-4) in DNA samples from the Buffy coat layer using conventional PCR (?). It may not be the best way to do it, but is it possible?
Thanks!
In genomic DNA you will only be able to detect the presence of the cytokine GENE in the samples so if the procedure is performed well you will have a 100% of positivity (uninformative).
For measuring transcription of the cytokine genes (secretion of the protein inferred) you would need RNA, cytokine mRNA that needs to be made (by retrotranscription) into cDNA, the PCR template. In other words, you need an RT-PCR (mRNA) not a conventional (DNA) PCR to perform what you seek. The RT-PCR can be done in a conventional thermal but this will give you only a semiquantitative measure. In regard to cytokine analysis, the aforementioned analysis can be useful to observe patterns of activation such as Th polarization (i.e. Th1 IFN-g high, IL-4, IL-10 low), especially if combined with transcription factors (T-bet, GATA3, RORgT, FoxP3, etc). In summary, NO, for detection of cytokines you need at least mRNA. For quantitative measures, you would need to perform quantitative PCR (i.e. real-time), of protein measuring techniques such as ELISA or flow cytometry. I am open to answer further questions on the topic. Cheers
Luis is correct. You need to collect mRNA and then reverse transcribe into cDNA and then do the real-time PCR (i would suggest you use the Taqman platform). Remember that you also need to select house keeping genes to normalize the data.
If you have never done any of these type of analysis, I would suggest you find someone at your institution who can help you.
Just to add that cytokines are determined using ELISA while molecular studies are for PCR
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