Can anyone help me in deciding which marker for expression analysis is better between immunohistochemistry and RT-PCR in FFPE tissues samples.
The Following text deals with tissue probes:
You can´t compare one IHC with one other RT-PCR but every IHC-Protein has also an RT-PCR-product (The RNA of the protein); e.g. AMACR for prostate cancer; see my publication from 2006).
The RT-PCR is objective and shows an average of the tissue containing some RNA and is not dependant from subjective interpretation of the pathologist.On the other side you have to deal with technical problems. Doing three RT-PCRs, you will receive three values and you have to take the average. RT-PCR is more expensive.
In Conclusion i think that RT-PCR can ad sensitivity and specifity to conventional IHC.
Thanks Christian and Martin. I was wondering if there is some effect of formalin fixation on the quality of RNA extracted as therefore, can IHC be a good option?
I think the answer also depends on what exactly your experimental questions are: IHC may give you important information on protein localization (ie, which cell the protein is expressed in, whether it is co-expressed with another protein, etc), RT-PCR will give you message abundance and is more easily quantifiable, but may not be indicative of protein abundance. Both have technical pluses and minuses...
Consider techniques that are totally different and the results are not fully comparable:
immunohistochemistry: The targets are proteins. Medium or low sensitivity with respect to qPCR. As a quantitative method is medium or low calidad.Te serves as a qualitative method but fine variations definitivbamente is not useful. The variability due to differences in reagent lot is high. The analysis results gives little information about pipetting error.
qPCR: Target is RNA. High sensitivity to immunohistochemistry. It is very good qualitative and quantitative method. You have to be more careful with the sample quality and control choice. I can give you some tips about it. Variations between batches of reagents are minor and depending on the brand of the equipment can be compensated easily. Allow to detect fine variations between the level of mRNA expression. The reliability of the result depends on the error due to the equipment (Roche LightCycler 2.0 has very little error), due to the reagent (TibMolbiol has worked very well), due to the sample (scanning from 200 to 300nm can identify many reaction inhibitors ). THE MOST IMPORTANT ERROR IS PIPETTING. This error is easy to identify by looking amPlification curves and melting curves.
I hope you find this information useful
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