The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display.
In many applications, an understanding of differentially expressed genes in different tissues or owing to an applied stimulus is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR [RAP-PCR] and differential display [DDRT-PCR]) has shown that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR, a technique has recently been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application. We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR) is a reliable and useful modification of available differential expression methods.
Kociok
N
N
Department of Vitreoretinal Surgery, University Eye Hospital, University of Cologne, German. norbert.kociok@uni-koeln.de
Unfried
K
K
Esser
P
P
Krott
R
R
Schraermeyer
U
U
Heimann
K
K
D002478:Cells, Cultured; D017242:Complement Factor H / Q000235:genetics; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis*
C493495:CFH protein, human; D020690:GTPase-Activating Proteins; D008869:Microtubule-Associated Proteins; D011506:Proteins; D012333:RNA, Messenger; C085284:microtubule-associated protein 1B; D017242:Complement Factor H