Improved differential screening approach to analyse transcriptional variations in organized cDNA libraries

Gene
E PerretD Caput

Abstract

A cDNA library was generated from rat brain tissues and organized into 1536-well plates, using a fluorescence activated cell sorter (FACS), acting as a single cell deposition system. The organized library containing 10,000 clones, with 60% full-length cDNA inserts, allowed the generation of multiple identical membrane replicas. Each replica was hybridized with a complex probe obtained from a particular brain tissue or a given cultured cell. The signal intensity for each of the clones present on the membrane, quantified with a standard image-analysis software, is proportional both to the abundance of the corresponding mRNA in the probe and to the amount of plasmid template on the membrane. The latter value was thus used to normalize the signals produced with complex probes, to optimize the comparison of mRNA expression levels for the different systems under study. The construction of high-quality cDNA libraries, the generation of identical membrane replicas and comparable probes, and the utilization of an image-analysis software package, coupled with the normalization of the spot intensity by assaying plasmid quantity, significantly improves the differential screening approach. Altogether, these technical improvements open the p...Continue Reading

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Citations

Sep 15, 2000·Journal of Immunological Methods·F L BattyeD M Tarlinton
Jun 6, 2002·Trends in Genetics : TIG·Robert Nadon, Jennifer Shoemaker
Dec 22, 1999·Genetic Analysis : Biomolecular Engineering·V GoffinP A Kelly
Aug 13, 2003·Plant Physiology·Fábio T S NogueiraPaulo Arruda

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