Technical Issues in Experimental Design

The sample preparation methods and the separation techniques have to be developed carefully in order to facilitate the entire biomarker discovery process. Sample preparation methods should be compatible with the subsequent steps in the sample preparation process. The use of a sample preparation method that results in the sample being in a solution that is incompatible with the separation method to be used will cause problems such as increased experimental difficulty, sample loss, increased time, and experimental cost. A good sample preparation method will facilitate quality results and the collection of good data. Good-quality results are easier to analyze and less prone to experimental errors that require high numbers of replicates to control. 

It is important to make sure that the methods used for sample preparation are reproducible and robust. It is not important at this stage to develop a method that will be used during validation and any subsequent clinical method. The goal of discovery work is to see as much of the sample diversity as possible. Remember that biomarker discovery is a statistical game where the more choices that you have the better the chance that you will find a marker that can be validated, so it is important to see as much of the diversity in the chosen class of molecule as possible. One should try and find a method that will eliminate classes of biomolecules that are not of interest, will interfere with the analysis of molecules that are of interest, complicate the separation/detection of the species of interest, and could cause confusion during data analysis. Time and effort spent in the process of sample preparation will make life much easier in subsequent experimental processes. 

The preparation of samples should focus on the type of molecule that one is interested in. Methods have been developed for the isolation of classes and subclasses of biomolecules these methods can be found in the literature and it is 

It is also worth some time to talk about the level of abundance of molecules. In general, the more abundant a molecule is in a system the less scientifically interesting the molecule tends to be. The reason for this is manifold, but one reason is that the tools for the analysis of compounds are all the same in one respect it is easier to see the most abundant species and thus they have been studied the most and a lot of information is available. Thus, the rarer a species the more scientific interest it generates since less is known about the molecule. In the case of biomarkers for medical applications, it is the ability to detect and quantify a marker that should be of primary concern. This makes more common molecules of more interest since they are easier to detect and quantify than compounds that are extremely rare. Furthermore, one can use a smaller sample size and simpler sample preparation procedures this becomes important when a large number of samples must be run for validation of a marker. 

It is also advisable to prepare tissue samples in a logical way based on the biochemistry of the molecules of interest and use tools such as subcellular fractionation to assist in the separation of your molecules of interest For example, if you are looking for changes that are occurring in the mitochondria, it is advisable to isolate these organelles and work with a pure mitochondrial sample rather than a sample contaminated with cytosol and other organelles since the simpler the sample the better your chances of seeing a wider spread of the diversity in the sample. 

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The search for biomarkers is not a simple task. There are several challenges in the pathway to a validated biomarker. These challenges include sample availability, the use of large numbers of samples for validation, technical issues in experimental design, assay development, the necessity for bioinformatics, and asking the right question so that the results of the experiment will have meaning and be of practical use in the clinic. 

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