PubMed · 10139359
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N E Trotto. 1994. Stat testing, Part 2. Stat testing triumphs and disappointments.. https://pubmed.ncbi.nlm.nih.gov/10139359/
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The aim of this study was to evaluate a direct and automated post-polymerase chain reaction (PCR) detection system to simultaneously determine the relative gene expression levels of nine cancer-related human genes. Total RNA was prepared from flash-frozen biopsies derived from human colorectal tumors or normal mucosa and reverse-transcribed to cDNA which was PCR-amplified using primer pairs corresponding to the studied genes. In each reaction, the forward primer was labeled with a fluorescent dye. The PCR products were pooled and an internal size standard with a uniquely colored fluorescent dye was added. The samples were then subjected to automated capillary gel electrophoresis. Fragment analysis software was used to calculate the relative gene expression using beta-actin as the reference gene. We found that automated capillary gel electrophoresis with multicolor detection is a rapid, accurate and highly reproducible method for separation and quantification of PCR-amplified cDNA.
An automated mass spectrometric linked fraction collection system is described which enables on-line examination of isolated components based on molecular-ion and product-ion information. This system has been used successfully to specifically isolate drug-related material from complex biological fluids to aid structural identification. Metabolites have been isolated with sufficient purity to allow unequivocal characterisation by 1H nuclear magnetic resonance spectroscopy. This system has the advantage that isolation of the components of interest is not triggered by a simple contact closure. Therefore fraction collection is not biased by limitations in either the detector (e.g. insufficient sensitivity) or the analyst (e.g. programmed collection of predicted metabolites only). Furthermore, all isolated components are readily available post fractionation for additional screening.
There is a continuing need for increased throughput in the evaluation of new drug entities in terms of their pharmacokinetic parameters. One useful parameter that can be measured in vitro using liver microsomal preparations is metabolic stability. In this report, we describe an automated system that can be used for unattended quantitative analysis of liver microsomal samples for a series of compounds. This system is based on the Sciex API 150 (single quadrupole) liquid chromatography/mass spectrometry system and utilizes 96-well plate autosampler technology as well as a custom-designed AppleScript which executes the on-line data processing and report generation. It has the capability of analyzing at least 75 compounds per week or 300 compounds per month in an automated fashion.