Multidimensional differential display via ion-pair reversed-phase denaturing high-performance liquid chromatography.
Experimental approaches are now available for the analysis of whole transcriptome expression in cells and tissues. Since the introduction of such methods for the investigation of differences in mRNA populations, they have been applied successfully to many areas of biology and medicine including development, differentiation, physiology, pharmacology, and carcinogenesis. Here we describe an improved and automated approach based on the differential mRNA display method developed by Liang and Pardee (P. Liang and A. B. Pardee, 1992, Science 257, 967-971). We report the use of ion-pair reversed-phase denaturing high-performance liquid chromatography (IP RP DHPLC), for the first time, to produce a "fingerprint," after amplification of the cDNA corresponding to the mRNA populations, from two or more of the samples that are to be compared. By overlaying the chromatograms produced from the amplification of different samples derived from the same set of oligodeoxynucleotide primers, those genes that are differentially expressed can be selected and subsequently cloned and sequenced rapidly to establish a profile of differentially expressed genes. In addition, validation of the data obtained is readily achieved by this method using IP RP DHPLC and quantitative RT-PCR. In this study total RNA was prepared from NTERA2 cells before and after differentiation induced by retinoic acid and was reverse-transcribed into cDNA prior to amplification to produce fluorescently tagged products. This methodology facilitates multiple rounds of interrogation of RT-PCR products and we tentatively refer to this approach as Multidimensional Differential Display.
Matin
Maryam M
MM
The Transgenomic Research Laboratory, Department of Molecular Biology, Krebs Institute, Firth Court, Sheffield, S10 2TN, United Kingdom.
Andrews
Peter W
PW
Hornby
David P
DP
D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger