Screening differentially expressed cDNA clones obtained by differential display using amplified RNA

Abstract

The major obstacle of differential display is not the technique itself but rather the post-differential display issueof discriminating between false positives and the truly differentially expressed mRNAs. This process is arduous and requires large amounts of RNA. We present and validate a method which allows one to screen putative positives from differential display analysis using only micrograms of total RNA. More importantly, we demonstrate that cDNA probes generated from amplified RNA are representative of the starting mRNA population and can be used for differential screening of mRNA species at a detectable limit of sensitivity of>/=1/40 000.

New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma

“…Vogeli-Lange et al 50 have introduced reverse-Northern analysis, which screens clones of DD-PCR products by hybridization with labeled original DD-PCR reactions. Poirier et al 51 have used amplified RNA to hybridize with the clone of the DD-PCR product. Other reported methods for screening differentially amplified cDNA products are by affinitycapturing of cDNAs or single-strand conformation polymorphism.…”

Altered Gene Expression: Induction/Suppression in Leek Elicited by Iris Yellow Spot Virus Infection (IYSV) Egyptian Isolate

“…DD-PcR provides a powerful method for the rapid identification of differentially expressed genes, and has been used successfully to identify such genes in both the host and the pathogen during infections (35). Benito et al (7) noted that it was difficult to prove to what extent the viral symptoms cause or are a consequence of such changes.…”

Immediate early gene X-1 (IEX-1), a hydroxytamoxifen regulated gene with increased stimulation in MCF-7 derived resistant breast cancer cells

“…To screen cDNA fragments cloned in pGEM-T vector for modified expression, we immobilized their PCR-amplified products on two identical Nylon membranes using a dot-blot apparatus and hybridized the membranes to [32] P-labeled cDNAs from either OHT sensitive MVLN or resistant MVLN OHT cells that had grown for 3 days in DCC medium and then for 4 days in DCC medium containing 10 −7 M OHT, according to Poirier et al [24]. Various quantities of ␤-actin PCR product were spotted on membranes to standardize signals.…”