Identification of differentially expressed mRNA species by an improved disply technique (DDRT-PCR)

Abstract

We have significantly improved a method originally developed by Liang and Pardee [Science 257 (1992) 967-971] to display a broad spectrum of expressed genes and to detect differences in expression between different cell types. We have analysed various aspects of the technique and have modified it for both, the application to fast and efficient identification of genes and the use with automatic analysis systems. Based on the mathematical background we have devised the appropriate number of optimal PCR primers. We have also introduced nondenaturating gels for separating double stranded fragments as single bands. By applying the method to regenerating mouse liver, we have identified, out of a total of 38,000 bands, about 70 fragments where the expression of the corresponding genes seems to be differentially regulated at different time points. Application of the method to an automatic DNA sequencer was successfully done. Thus, we have confirmed the usefulness and increased the power of the RNA display technique, which we named differential display reverse transcription PCR (DDRT-PCR), and have extended the range of its application.

Wig-1, a new p53-induced gene encoding a zinc finger protein

“…PCR-based di erential display was performed as described (Liang and Pardee, 1992;Liang et al, 1994;Bauer et al, 1993), with the following changes: 30 mg of each total RNA was treated with 30 U DNase I (GIBCO-BRL, MD) at room temperature for 15 min, extracted with phenol/ chloroform twice and chloroform once, and precipitated with ethanol. After suspention in DEPC-treated dH 2 O, 0.4 mg of RNA were reverse-transcribed in a total volume of 20 ml containing either 1 mM T 13 A, T 13 C or T 13 G, 20 mM dNTPs, 10 mM DTT, 16RT bu er (25 mM Tris-HCl, pH 8.3, 37.5 mM KCl, 1.5 mM MgCl 2 ) and 200 U Superscript II reverse transcriptase (GIBCO-BRL, MD) for 60 min at 408C.…”

SAGE transcript profiles for p53-dependent growth regulation

“…Recent developments in technologies aimed at identifying di erentially expressed genes have yielded valuable insights into the mechanisms governing cell growth control (Liang and Pardee, 1992;Bauer et al, 1993;Lee et al, 1995). Although numerous genes have been identi®ed, the limited and random data acquisition from these technologies preclude a comprehensive evaluation of all genes involved in cell growth regulation.…”

Sequence analysis and exclusion of phosducin as the gene for the recessive retinal degeneration of the abyssinian cat

“…Phosducin is a highly conserved 33 kDa phosphoprotein that was initially found in the rod and cone photoreceptors, as well as in the pineal gland [4], but is now recognized to be present in other tissues [5,6]. This soluble protein is distributed within the cytoplasm of the photoreceptors.…”