Methods to prepare RNA and to isolate developmentally regulated genes from Eimeria.
Coccidians represent a large class of important intracellular parasites that traverse multiple developmental stages that are distinct and required to complete the life cycle. The biochemical details underlying the regulation of transformation from one developmental form to the next are limited and the study of such details presents unique obstacles. However, the genetic program is critical and may provide a basis for understanding the biology of these organisms in addition to the opportunity to suppress development and infection. We provide a basic overview of several strategies, including previously unpublished results, used by this laboratory to isolate stage-specific genes from Eimeria bovis. Additionally, we have included detailed discussions that summarize the associated advantages and disadvantages of each as applied to coccidia and potentially to other parasites in the phylum Apicomplexa. Given that the purification of sufficient quantities of high-quality RNA is vital, we have included detailed protocols for the isolation of RNA from various parasite stages. Also included is a detailed protocol to apply mRNA differential display to investigate stage-specific developmental regulation.
White
M W
MW
Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, 59717-0360, USA. uvsmw@gemini.oscs.montana.edu
Radke
J R
JR
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary; D004539:Eimeria / Q000235:genetics* / Q000254:growth & development; D018507:Gene Expression Regulation, Developmental; D017125:Genes, Protozoan; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D016053:RNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D016053:RNA, Protozoan; D012194:RNA-Directed DNA Polymerase