Laser capture microdissection of Plasmodium falciparum liver stages for mRNA analysis.
Plasmodium falciparum liver-stages are important targets for vaccine-induced protective immune responses and prophylactic treatment against malaria. Little is known of the gene expression profile of malaria parasites during their development inside hepatocytes. The sequencing of the P. falciparum genome and the development of DNA microarray technology give new opportunities to identify genes expressed during the development of Plasmodium. However, transcriptome analysis cannot currently be applied to the hepatic stages, due to difficulties in obtaining sufficient amounts of parasite material that lie among the large excess of host cell RNA. Here, we describe the isolation of liver-stages by a modified laser capture microdissection approach applied to human hepatocyte cultures infected with P. falciparum. RT-PCR amplification of several P. falciparum transcripts demonstrated the high quality of the RNA recovered after microdissection. This approach should enable analyses of P. falciparum transcriptome during its hepatic development and substantially assist the identification of new therapeutic and vaccine targets.
Semblat
Jean-Philippe
JP
INSERM, Unité U511, Immunobiologie Cellulaire et Moléculaire des Infections Parasitaires, CHU Pitié-Salpêtrière, Université Pierre et Marie Curie, 91 Boulevard de l'Hôpital, 75013 Paris, France.
Silvie
Olivier
O
Franetich
Jean-François
JF
Hannoun
Laurent
L
Eling
Wijnand
W
Mazier
Dominique
D
D000818:Animals; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology / Q000469:parasitology*; D006801:Humans; D007834:Lasers; D010963:Plasmodium falciparum / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D016053:RNA, Protozoan / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan