Abstract
BackgroundRecent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes.Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures.ResultsIn the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube® format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well.ConclusionAs the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.
Highlights
Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes
Ney) 293 (ACC # 305) cells according to three different methods: A) Labelling of reverse transcribed cDNA in the course of the reaction with Klenow fragment, B) Labelling with Klenow fragment after amplification of total RNA according to the Eberwine protocol [19] and C) synthesis of double stranded cDNA starting from total RNA and labelling in the course of linear amplification using a socalled single primer amplification (SPA)-primer
The question was whether the improvement achieved by the SPA method was subject to a better signal sensitivity, to a better signal specificity or to both
Summary
Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. The Cathepsins B, L, K and S may play an active role in cancer development [8] and inflammation diseases like rheumatoid arthritis [9]. Because of the importance of proteolysis in vivo, it is of great interest to better understand how the balance between active proteolytic enzymes and their endogenous inhibitors is controlled. The introduction of synthetic inhibitors of proteinases as therapy concepts drew high expectations for controlling or limiting the degradative potential of proteinases in e.g. cancer or rheumatoid arthritis [9]. Expression analysis using DNA microarray technology is one potent tool to study the involvement of proteases in various normal and pathological processes or to follow the expression during an inhibitor treatment. It must be urgently stressed that such analyses require confirmation by independent methods such as e.g in vivo activity measurements of enzymes or at least QRT- PCR measurements
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