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Description:
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Gene expression microarray technology is potentially capable of examining all of the cellular processes at the mRNA level at a given moment . One major challenge in its applicability is that most biological samples are cell mixtures and the cell type of interest is often a minor cell subset . Using well -defined mixtures of model cell types with different cell ratios we found that the overall gene expression profile (GEP ) of mixed cell populations was the weighted average of the GEPs for each cell subpopulation in the cell mixture . Thus , without applying any cell separation the cell type in majority dominated the overall GEP of the sample while the GEPs of minor cell subsets were diminished . We showed that the functional threshold for the necessary purity of a cell type in a sample to produce virtually identical overall GEP to a pure sample was 75 % and this could be achieved by conventional cell sorting methods without altering the overall GEP in the process . For the purification of small , biohazardous samples , we tested the applicability of multistage magnetic sorting (Magsort ) and laser enabled analysis and processing (LEAP ) . We developed optimized sample labeling and sorting protocols for both technologies and demonstrated that while the maximum purity we could achieve with Magsort was 75 -80 % , with the LEAP instrument we could purify fluorescently labeled cell subsets to 80 -100 % . The purified cells from biological samples often do not provide enough RNA for direct microarray studies without RNA amplification . We found that both linear and exponential amplification was capable of producing enough RNA for microarray analysis even from a single cell . Both methods distorted the GEP , however , with linear amplification much fewer genes were affected and only this method preserved the GEP differences between samples . Further studies are needed to analyze and possibly eliminate all GEP distortion . In conclusion , the purification of minor cell subsets from biological samples prior to microarray analysis is not only necessary , but also achievable without GEP distortion . Using linear RNA amplification of small purified samples , meaningful microarray data can be produced about the GEP of even a few cells . |