Thanks for the information.

There are several things I would like to add:

The easiest way to check your experimental setup (also GMP!) would be positive (negative) controls. Use a well know and described EGFR inhibitor to see if everything is working fine and the inhibitor is showing the desired effect in your system compared to EGF stimulation alone. Then you know if you have a problem with the primers or with the whole setup…

PCR:

I still have a few questions concerning the PCR: So you are running PCRs for H-Ras and ElK1, right? And you are doing qRT-PCR? With SYBR or Taqman? Do you normalize the data against a reference gene (if yes, which?). If you want to detect changes it’s important to run an internal control to see if you are using the same amount of template.

Cells and inhibitor:

Yes, it’s crucial (!!!!) to serum starve the cells prior to stimulation to achieve a manageable basal level of activation (and thus to be able to see the effect of a single growth factors). If the cells tolerate it well I would even suggest lengthening the time to 4h.

Do you know the Kd of your inhibitor (or how long it is stable in medium?)? I would add it at least 15min before adding EGF to your samples. Are you using the right concentration?

I would also strongly advise you to do a time course to better detect the changes. 24h is quite long for such an experiment (mRNA changes can normally be observed after 10min+) with a novel and undescribed (I presume it is one?) inhibitor. I would try 15min, 30min, 1hr, 2hr and 4hr.

Also Kunal Pratap Singh added a good point. IF you are using a novel inhibitor (which supposedly binds to EGFR?), are you really sure it’s not agonist? Have you (or anyone else) done preliminary tests (like ERK activation) for prove of concept?

Your welcome :)

Hello Veronica,

It would be very helpful to provide more information about your experiment. Like: What target are you analyzing? What is the formulation of your media? How long is your stimulation time?

Since different feedback inhibitory mechanisms restrain EGFR activity in time changes of certain targets on mRNA level can be tricky to demonstrate.

Just a general thought:

You could also check for a more direct effect of EGF stimulation like phosphorylation of the EGF-R or activation of downstream targets like Ras or ERKs.

Thank you so much for the suggestion.

I am interested in EGFR inhibitor by following the inhibition in the expression of the cascade as H-Ras (Inner membrane) and ElK1(Nucleus).

In the beginning I was using 10%FBS + 1% P/S in my RPMI 1640 Media by adding EGF at the moment of the stimulation.

Now I am given first 2h of serum starvation, according to one professor research said that FBS had some growth factor and can interfered with my stimulation (Mostly I was discussing about Western Blot and p-EGFR) according to this I start after give the time of serum starvation, the treatment and stimulation at the same time by using Free serum RPMI 1640 + EGF and Inhibitor for 24 hours, after this I isolate the RNA by Tryzol technique.

I actually not sure if my problem is with the stimulation of EGF or primer, but I think it is not primer design problems because I try with 3 different and all with some intensity different are giving me the opposite data that I expect.

I hope this information can help you to help me. Thank you so much!

Thanks for the information.

There are several things I would like to add:

The easiest way to check your experimental setup (also GMP!) would be positive (negative) controls. Use a well know and described EGFR inhibitor to see if everything is working fine and the inhibitor is showing the desired effect in your system compared to EGF stimulation alone. Then you know if you have a problem with the primers or with the whole setup…

PCR:

I still have a few questions concerning the PCR: So you are running PCRs for H-Ras and ElK1, right? And you are doing qRT-PCR? With SYBR or Taqman? Do you normalize the data against a reference gene (if yes, which?). If you want to detect changes it’s important to run an internal control to see if you are using the same amount of template.

Cells and inhibitor:

Yes, it’s crucial (!!!!) to serum starve the cells prior to stimulation to achieve a manageable basal level of activation (and thus to be able to see the effect of a single growth factors). If the cells tolerate it well I would even suggest lengthening the time to 4h.

Do you know the Kd of your inhibitor (or how long it is stable in medium?)? I would add it at least 15min before adding EGF to your samples. Are you using the right concentration?

I would also strongly advise you to do a time course to better detect the changes. 24h is quite long for such an experiment (mRNA changes can normally be observed after 10min+) with a novel and undescribed (I presume it is one?) inhibitor. I would try 15min, 30min, 1hr, 2hr and 4hr.

Also Kunal Pratap Singh added a good point. IF you are using a novel inhibitor (which supposedly binds to EGFR?), are you really sure it’s not agonist? Have you (or anyone else) done preliminary tests (like ERK activation) for prove of concept?

Your welcome :)

Thank you so much for all the suggestions.

I am would like to observe the effect of my "inhibitor" (Is new and natural isolated compound that works for other proteins and gene but now I am testing for EGFR pathway), by molecular docking we observe the possible binding with the EGFR and H-Ras proteins compering with a control drug. The prediction data show that maybe can have good activity, therefor I go now for in vitro.

Actually, I am now doing Reverse Transcriptase PCR. For my drug activity I also start to think can maybe be an agonist by actually may main concern is only by observing the control (no stimulated) and positive control (EGF stimulated) the density of the bands is less for the positive control, that why I was thinking the stimulation can be grown. With the previous sample, in which I use complete media for my treatment and stimulation, I was did qRT-PCR. In that time I use SYBR and as housekeeping gene B-Actin. But, the results wasn't significant in that moment.

I really don't know the Kd of my inhibitor, but previous experiment commonly use for treatment from 12 to 48 hr. I also use in that range for experiment not related with gene expression. But, exactly I don't know the  stability of my compound.

There is one paper in which they show that my compound can decrease the expression of p-ERK (western blot) after stimulate with TNF-alpha using different cell line. But, I don't have more information about the activity with ERK.

By following your previous suggestion I will try some new stimulation steps, and again really thanks for you help.

I agree that Nina's suggestion of a time-course may well help you.  Because of feedbacks, a stimulation can sometimes be followed by a dip below baseline, and then a rise again (damped oscillation) before steady-state.  That might explain why you see a dip in the response with EGF itself at the single time of 24 h. 

Does EGF produce a biological response in your cells, like stimulating cell division?  If so you could also test for inhibition of that.  (If not, then maybe it would be better to use a cell type that does have a positive biological response.)

Hi Veronica

I know qRT-PCR is pricey and can be difficult to analyse, but normal RT-PCR is in most cases really NOT suited to examine expression differences (unless you have a really strong activation or reduction (500% or something)).…

Do I get the point right, that you see a less intense band in your EGF-stimulated sample compared to your unstimulated sample after 24h? With all the feedback loops of prolonged EGF-R signaling this can actually be physiological.  So shorten the stimulation time und check with qRT-PCR.

I think the results from your previous qRT-PCR were not significant because you used complete medium. Also, PLEASE do yourself a favor and change the housekeeping gene. Actin (like most cytoskelatal proteins) is more than often not stable when using growth factors or cytokines as stimulants. Consider using two instead of one housekeeping genes right from the start. That will make publications in good journals later on more easy (for more information: google MIQE guidelines).

If you have the equipment to do western blot for p-ERK I would strongly advise you to do so. It's a two day experiment, the available antibodies work really well and the results are trusted in the scientific community.

Good luck with your next experiments.

Thank you so much for all  your help. Now I am doing in different why following all your advice. I hope to have good result.