Molecular and Biochemical Parasitology, 4 (1981) 337-348 337 Elsevier/North-Holland Biomedical Press PREPARATION AND IN VITRO TRANSLATION OF mRNA FROM FASCIOLA HEPATICA DAVID O. IRVING and MICHAEL J. HOWELL Department o f Zoology, Australian National University, Canberra, A. C. T. 2600, Australia (Received 25 June 1981;accepted 28 September 1981) Total RNA was extracted from mature and juvenile Fasciola hepatica by homogenizing in 5.0 M guanidine thiocyanate and centrffugation through a 5.7 M CsC1 cushion. Yields of 2 mg/g and 1 mg/g wet weight starting material were obtained, respectively. Messenger RNA wasseparated from the bulk extracted RNA by binding to oligo(dT)-cellulose. About 25% of the extracted RNA bound in both adult and juvenile cases. This material was subsequently tested in a rabbit reticulocyte cell-free trans- lation system. Up to a 12-fold stimulation of incorporation of [3SS]methionine into trichloroacetic acid-precipitable material was observed over that where no message was added. When the in vitro translation products were analysed by autoradiography of SDS-polyacrylamide gradient gels, poly- peptides ranging in apparent molecular weight from about 10 000 to 100 000 were observed. Several minor differences in the electrophoretic patterns obtained from juvenile and adult mRNA were ob- served. Key words: Fasciola hepatica, Excretory- secretory antigens, mRNA, In vitro translation, SDS-polyacrylamide gel electrophoresis. INTRODUCTION Attempts to vaccinate sheep against infection with the liver fluke, Fasciola hepatica, have been unsuccessful [1,2]. In a number of studies, homogenates of whole flukes have been used as vaccines. That these are ineffective could be due to the complexity of the homogenate in terms of the number of proteins present. Any antigen which has the potential to evoke a protective host response may be present in concentrations too low to be of any significance. Moreover, many of the proteins in the homogenate probably never come into contact with the host during the course of a natural infectioh, and would, therefore, be inconsequential in terms of stimulating any protective response. Abbreviations: AMV, avian myeloblastosis virus; DEP, diethyl pyrocarbonate; ES antigens, excretory- secretory antigens; NTE buffer, 0.1 M NaC1, 10 mM Tris-HC1 (pH 7.6), 5 mM EDTA; oligo (dT)- cellulose, oligothymidylic acid cellulose; PBS, phosphate-buffered saline; poly(A+)-RNA, polyadenyl- ated RNA; SDS, sodium dodecyl sulphate; TE buffer, 10 mM Tris-HC1(pH 7.6), 5 mM EDTA. 0166-6851/81/0000-0000/$02.75 ©1981 Elsevier/North-Holland Biomedical Press