Laser-assisted microdissection: a new tool for aquatic molecular parasitology.
Laser-assisted microdissection (LMD) has been developed to isolate distinct cell populations from heterogeneous tissue sections, cytological preparations, or live cell samples. Downstream applications typically include gene expression studies using real-time PCR and array platforms, diagnostic PCR, and protein expression studies. LMD techniques are now commonplace in mainstream biological research and clearly have suitable applications in the field of aquatic pathology and parasitology. The present study used LMD to isolate 2 dinoflagellate parasites (Hematodinium spp.) from formalin-fixed paraffin-embedded tissue sections from 2 crustacean hosts, Cancer pagurus and Portunus trituberculatus. DNA was isolated from LMD parasite preparations, and partial regions (up to 300 bp) of the small subunit and the first internal transcribed spacer region of the rRNA gene complex from the Hematodinium spp. were PCR amplified using diagnostic primers. The amplification products were sequenced to confirm the identity of the targeted regions. The techniques, applications, and limitations of LMD to address questions in aquatic molecular pathology and parasitology are discussed.
Small
H J
HJ
Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth, Dorset DT4 8UB, UK.
Sturve
J
J
Bignell
J P
JP
Longshaw
M
M
Lyons
B P
BP
Hicks
R
R
Feist
S W
SW
Stentiford
G D
GD
D000818:Animals; D003386:Brachyura / Q000469:parasitology*; D021901:DNA, Intergenic / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000302:isolation & purification; D004141:Dinoflagellida / Q000166:cytology*; D043143:Hepatopancreas / Q000469:parasitology; D007834:Lasers; D042282:Microdissection / Q000295:instrumentation* / Q000379:methods*; D018482:Muscle, Skeletal / Q000469:parasitology; D016133:Polymerase Chain Reaction
D021901:DNA, Intergenic; D016054:DNA, Protozoan