mRNA differential display of acute-phase proteins in experimentalEscherichia coli infection

Abstract

We present a modification of mRNA differential display in which increased throughput results from the use of an automated fluorescent sequencer. The sequence analysis is performed directly on purified fragments without further cloning. The amplified fragments carry a T7 RNA polymerase promoter sequence tag for in vitro transcription of riboprobes for nonradioactive in situ hybridization. We compared changes in gene expression in the liver and colon of group II phospholipase A2 transgenic and group II phospholipase A2 deficient mice during the course of experimental Escherichia coli infection. Fluorescent mRNA differential display comprising a 7 x 24 set of primers was used to study a total of 31,257 amplified cDNA fragments. Sequence analysis of the displayed fragments associated with infection identified classical acute-phase proteins in the liver and host defense proteins in the colon. The displayed mRNAs associated to transgenicity were the transgene itself, i.e., human group II phospholipase A2, and glutathione-S-transferase in the liver. In the colon, the displayed mRNAs associated with transgenicity were the pancreatitis-associated protein and mucin. The results show that fluorescent mRNA differential display is a reliable method to identify differences in the expression of the genes of acute-phase proteins.

mRNA differential display of gene expression in colonic carcinoma

“…Six RNA samples were each reverse transcribed to 15 different cDNA pools by the use of the three possible combinations of one base anchored and the twelve possible combinations of two base anchored cDNA primers as described earlier [16]. The anchored primers (synthesized by APBiotech, London, UK) shared a common 5'-design, a T7 sequence tag (underlined) followed by dT 14 , (5' TAC-GACTCACTATAGGGAGTTTTTTTTTTTTTT.…”

“…Three micrograms of RNA and 75 pmol of RT-primer were pipetted into the wells of a 96-well polypropylene PCR plate (Costar, Cambridge, MA, USA). The cDNA reaction conditions using Superscript II (Gibco BRL Life Technologies, Rockville, MD, USA) and Hybaid express thermocycler (Hybaid, Middlesex, UK) were as described earlier [16]. Prior to storage on GeNunc strips (Nunc, Roskilde, Denmark) at -707C, the reverse transcription reaction was stopped by heating at 707C for 10 min.…”

“…The differential display PCR using a common secondary T7 sequence tagged Cy5-labeled primer (Cy5-T7: 5' Cy5-GTAATACGACTCACTATAGGG 3'; APBiotech) as a downstream primer and a set of gene-specific primers as upstream primers was done as described earlier [16]. In addition to the described set of DD5'-1 to -24, 8 new primers (named DD5'-25 to -32) were used in the current study: The primers are complementary to rat group II phospholipase A2 gene (DD5'-25 and -26), human group V (DD5'-27 and -28) and group X (DD5'-29 and -30) phospholipase A2 genes and to a linker sequence (DD5'-31 and -32).…”

“…The fluorescently labeled PCR products were analyzed by electrophoresis through a 0.5 mm thick denaturing 4%T/3%C polyacrylamide gel with a separation distance of 20 cm (standard cassette) in an automated sequencer (ALFexpressII, APBiotech) as described earlier [16]. An internal Cy5-labelled size standard series of 633 bp, 647 bp and 680 bp was added to the electrophoretic samples to aid in size determination and evaluation of the occurrence of lane wandering.…”

“…However, this cannot be applied to automated fluorescent differential display. In our previous work [16], fragments were purified from a secondary electrophoretic agarose gel. The limitations of resolution of that approach have been circumvented in the current study by the use of secondary PAGE also for the fragment purification.…”