Preparation and in vitro translation of mRNA from Fasciola hepatica.
Total RNA was extracted from mature and juvenile Fasciola hepatica by homogenizing in 5.0 M guanidine thiocyanate and centrifugation through a 5.7 M CsCl cushion. Yields of 2 mg/g and 1 mg/g wet weight starting material were obtained, respectively. Messenger RNA was separated 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 translation system. Up to a 12-fold stimulation of incorporation of [35S]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, polypeptides 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 observed.
D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002845:Chromatography; D005210:Fasciola hepatica / Q000235:genetics* / Q000254:growth & development; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D011061:Poly A / Q000235:genetics* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C021722:oligo(dT)-cellulose; D011061:Poly A; D002482:Cellulose