Hi,

As you know, qPCR measures gene expression (mRNA level), with ELISA/Luminex measures protein concentrations. mRNA might not be translated to the proteins, so having protein concentration is a bit more biologically meaningful/accurate. On the other hand, it is sometimes hard to find ELISA/Luminex kits for specific chemokine and primers are easily available and considerably cheaper compared to ELISA/Luminex. You should also take into account that special instruments are required for both qPCR and Luminex (and are they are available to you?) and that the sample preparation procedures are not the same and it depends which one you are more familiar with (by the way, protein extraction from the tissue is cheaper in our hands than using RNA extraction columns).

Recently, we measured a bunch of chemokines (>20) with qPCR in our samples, and confirmed some interesting ones (5-6) at protein level (samples from different biological experiments, though, but this just adds to the reliability of the measurements, I think).

I hope this helps.

Cheers,

Berislav

Hello, I think you have to evaluate what is the question to want to answer, If you are assessing tissue chemokine expression, are you interested in local effect, systemic or both? chemokine mRNA expression can help more if you are looking for the first one. Also, as Berislav mentioned, not all the tissue/samples can be processed for measuring mRNA expression or protein concentration. Maybe if you point out which cells or tissues you are interested in, we could provide some tips yo you.

Cheers,

Marcela

If you have a culture methodology for the tissues you mention then Luminex is quite easy to measure many differnt cytokines secreted into the supernatant. Kits are available for most of the popular cytokines. If you only have biopsy type samples then some sort of RNA extraction might be the way to go with PCR as your end point measure. I have used both and find Luminex is easier to perform consistently. It is easier to acquire a supernatant in a reliable manner than an RNA extraction in my opinion. In the end culture, or growth condiions may account for most of the variability you see no matter what method you choose.

You mention "tissues infected with different cell lines". Would you be able to further elaborate?

Thank you very much for your replies. I am wanting to know whether there is an increase (or upregulation) in any chemokines at the site of infection (which in this case is the mouse ear infected with different Leishmania mutants) that could account for an increase in inflammatory monocytes.

Since you will be acquiring samples from tissue biopsy I would recommend using qPCR. Take care in developing control primers so you can determine relative quantities/copies of some other gene that you expect to be expressed at constant levels with or without Leishmania infection. This will allow you to control sample preparation. I think sample prep will contribute significantly to any variation you see and so will have to be adequately controlled for.

If you try a direct measure of cytokines from your biopsy, or a culture method than you could use an EIA based measure. I still think sample prep will be an issue and you will have to develop good controls. Also, sensitivity may be an issue too as you just may not have much cytokine to measure.

James

James

We have successfully used Luminex technology to determine cytokine and chemokine concentrations in mouse ears during cantharidin-induced inflammation (see Ivetic-Tklacevic et al. Transl Research 2012, 160:137). On the other hand, isolating good-quality RNA from skin samples proved to be rather difficult: we could not get RNA samples with RIN number better than 7.5.