High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

BACKGROUND: Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti. RESULTS: Total RNA was isolated from Ae. aegypti midguts that were either fresh-frozen or fixed with histological fixatives. Generally, fresh-frozen tissue sections are a common source of quality LMM-derived RNA; however, our aim was to develop an LMM protocol that could inactivate pathogenic viruses by fixation, while simultaneously preserving RNA from arbovirus-infected mosquitoes. Three groups (10 - 15 mosquitoes per group) of female Ae. aegypti at 24 or 48-hours post-blood meal were intrathoracically injected with one of seven common fixatives (Bouin's, Carnoy's, Formoy's, Cal-Rite, 4% formalin, 10% neutral buffered formalin, or zinc formalin) to evaluate their effect on RNA quality. Total RNA was isolated from the fixed abdomens using a Trizol(® )method. The results indicated that RNA from Carnoy's and Bouin's fixative samples was comparable to that of fresh frozen midguts (control) in duplicate experiments. When Carnoy's and Bouin's were used to fix the midguts for the LMM procedure, however, Carnoy's-fixed RNA clearly showed much less degradation than Bouin's-fixed RNA. In addition, a sample of 5 randomly chosen transcripts were amplified more efficiently using the Carnoy's treated LMM RNA than Bouin's-fixed RNA in quantitative real-time PCR (qRT-PCR) assays, suggesting there were more intact target mRNAs in the Carnoy's fixed RNA. The yields of total RNA ranged from 0.3 to 19.0 ng per ~3.0 × 10(6 )μm(2 )in the LMM procedure. CONCLUSIONS: Carnoy's fixative was found to be highly compatible with LMM, producing high quality RNA from Ae. aegypti midguts while inactivating viral pathogens. Our findings suggest that LMM in conjunction with Carnoy's fixation can be applied to studies in Ae. aegypti infected with arboviruses without compromising biosafety and RNA quality. This LMM method should be applicable to other mosquito vector studies.