Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, Louisiana 70112, USA.
Parasit Vectors. 2011 May 19;4:83. doi: 10.1186/1756-3305-4-83.
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.
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) μm2 in the LMM procedure.
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.
激光显微切割显微镜（LMM）具有作为研究工具的潜力，因为它可以从复杂的生物样本中精确切除目标组织或细胞，并促进组织特异性样品制备。然而，迄今为止，这种方法尚未用于蚊子载体。为此，我们开发了一种使用埃及伊蚊分离中肠 RNA 的 LMM 方法。
使用埃及伊蚊从中肠组织中分离出总 RNA，这些组织要么是新鲜冷冻的，要么是用组织固定剂固定的。通常，新鲜冷冻的组织切片是 LMM 衍生 RNA 的常见来源；然而，我们的目的是开发一种 LMM 方案，该方案可以通过固定来灭活致病性病毒，同时保持感染虫媒病毒的蚊子的 RNA。三组（每组 10-15 只蚊子）经胸部注射 7 种常见固定剂（Bouin's、Carnoy's、Formoy's、Cal-Rite、4%甲醛、10%中性缓冲甲醛或锌甲醛）中的一种，以评估其对 RNA 质量的影响。使用 Trizol®方法从固定的腹部中分离总 RNA。结果表明，在重复实验中，Carnoy's 和 Bouin's 固定剂样本的 RNA 与新鲜冷冻中肠（对照）相当。然而，当 Carnoy's 和 Bouin's 用于固定 LMM 程序中的中肠时，Carnoy's 固定的 RNA 明显显示出比 Bouin's 固定的 RNA 更少的降解。此外，在定量实时 PCR（qRT-PCR）测定中，使用 Carnoy's 处理的 LMM RNA 比 Bouin's 固定的 RNA 更有效地扩增了 5 个随机选择的转录本，这表明 Carnoy's 固定的 RNA 中存在更多完整的靶 mRNA。在 LMM 程序中，总 RNA 的产量范围为每 3.0×10(6)μm2 从 0.3 到 19.0ng。
发现 Carnoy's 固定剂与 LMM 高度兼容，可从埃及伊蚊中肠中产生高质量的 RNA，同时灭活病毒病原体。我们的研究结果表明，LMM 联合 Carnoy's 固定可应用于感染虫媒病毒的埃及伊蚊研究，而不会影响生物安全性和 RNA 质量。这种 LMM 方法应该适用于其他蚊子载体研究。
