Normal/Disease-Paired cDNA Library Subtraction for Molecular Marker Development

Abstract

This study consists of a novel strategy for the identification of potential cancer exclusively expressed genes, which might lead to the development of valuable diagnostic molecular markers. Normal- and cancer-paired tissues from the same patient were collected and subjected to the construction of primary complementary deoxyribonucleic acid (cDNA) libraries. The cancer cDNA library was generated by subtracting normal cDNAs from the primary cancer library. The remaining clones in the subtracted cancer library were sequenced and Basic Local Alignment Search Tool (BLAST)-analyzed against current nonredundant and est_human databases in the GenBank. The clones that had no matches with any known gene sequences except the human genomic sequences were identified as ideal candidates for potential molecular marker development. The candidate marker sequence and associated primer sequences were identified on the target clone sequence of the region with the least, or no, matched homologs. The specificity of the markers was measured by a polymerase chain reaction test experiment with the DNA templates from different human normal tissues, genomic DNA, and additional patients' tissues. Results from bioinformatical and experimental approaches used suggest that the methodology has a high potential to identify cancer exclusive transcripts. Thus, it might result in the development of diagnostic markers.

Mouse brain full‐length cDNA library construction by negative selection of intact mRNAs

“…Total cellular RNA was isolated from mouse C57BL/6J brain (male) by modification of a standard procedure [4,5]. Briefly, 1 g tissue was ground in liquid nitrogen and homogenized in 5 mL solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol) using a Polytron PT3000 homogenizer (Brinkmann) followed by addition of 500 μL 2 M sodium acetate pH 4.0.…”

“…The mouse brain standard cDNA library and fulllength cDNA library were constructed using our previously reported method [4][5][6]. Purified regular mRNAs (1 μg) and unknown amount of purified full-length mRNAs were primed by adding 200 ng biotinylated oligo-dT primer with an integrated…”

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

“…It is potentially a useful tool for gene expression profiling in new species [2], enhancing our understanding of many organismal molecular and physiological mechanisms [3-5], and facilitating the development of diagnostic markers and therapeutic drugs [6,7]. Most current protocols or commercially available kits apply relative subtraction, which is primarily based on the transcript variance of differentially expressed genes among investigated materials [7-9].…”

“…This method takes into account the challenges described earlier while simplifying the whole subtraction process. It was inspired from our previously reported paired library absolute subtraction technology [6] and is currently applied on our ongoing peanut ( Arachis hypogaea L.) genomics research. In this investigation, we had hypothesized identifying target genes expressed only in peanut immature pod by subtracting, in a single round, all expressed genes common to both tissues.…”