Automation of Fluorescent Differential Display With Digital Readout

Abstract

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Most of previous DD work has taken a "shot-gun" approach of identifying one gene at a time, with a limited number of polymerase chain reaction (PCR) reactions set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a new platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

The Transcriptomic Toolbox: Resources for Interpreting Large Gene Expression Data within a Precision Medicine Context for Metabolic Disease Atherosclerosis

“…Likewise, the other common routine hybridization technique, Differential Display, is a PCR-based method that also detects and measures differential gene expression without using specific primers, making it a robust, inexpensive discovery tool [16]. Current innovations incorporate the use of fluorescent labels with automation to yield high throughput analyses [17,18]. These techniques have been applied successfully to understand molecular and cellular pathways involved in stem cell differentiation by identifying the expressed genes causing lineage commitment into megakaryocytes, erythrocytes, and granulocytes, which play different roles in atherosclerosis disease developmental and disease progression [19].…”

Transcriptome profiling of lactococcal mixed culture activity in milk by fluorescent RNA arbitrarily primed-PCR

“…However, if the genome has not been sequenced, the techniques of differential display [30,31] can be applied to evaluate modifications in transcriptomes according to various conditions either in eucaryotes [10,29] or in procaryotes [32]. The two molecular techniques of cDNA amplified fragment length polymorphism (cDNA-AFLP) [1] and RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) [32] are the most frequently used for evaluating modifications in transcriptomes [15]. However, cDNA-AFLP requires restriction sites so transcripts without correct sites will not be revealed at all, while RAP-PCR uses acrylamide electrophoresis that lacks the resolution necessary to efficiently separate amplimers [2].…”