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RNA from LCM Samples

Juanita Gonzales, Ambion, Inc.

Obtaining specific populations of cells from heterogenous samples is a major challenge in studying gene expression profiles of specific cell types. Laser capture microdissection (LCM), developed in a collaborative effort between the NIH and Arcturus, Inc. specifically to isolate cancer cells from normal tissue, can be used to separate individual cell types from within complex tissues. RNA can then be extracted from these microdissected cells to analyze their gene expression.

How LCM Works
LCM can capture cells or discrete morphological structures from thin tissue sections. Frozen, O.C.T.-embedded tissue sections are prepared for the LCM process by fixation, staining, and dehydration. Sections are visualized through a thermoplastic film attached to the bottom of a microfuge tube cap. A laser pulse is directed through the film onto the target cells. The plastic film melts onto the targeted area, then cools and bonds with the underlying cells. The film, along with the adhered target cells, is collected (Figure 1). RNA can be isolated from these captured cells for real-time PCR and mRNA expression profiling. (See our tips article Getting Intact RNA from LCM Samples.)

Capture and Analyses of Dentate Gyrus from Mouse Brain
LCM was used to separate dentate gyrus from mouse brain sections (Figure 1). RNA from the captured dentate gyrus was purified using the RNAqueous-Micro Kit (see sidebar) and the purified RNA was assessed on an Agilent bioanalyzer (Figure 2). This RNA was of sufficient quality and amount for real-time RT-PCR analysis, which showed consistency between replicates and very low DNA contamination in the RNA samples purified with RNAqueous-Micro Kit (Figure 3).

Figure 1. LCM Collection of Dentate Gyrus from Mouse Brain. (A) Cryosection (10 micron) stained with cresyl violet. (B) Same section after Laser Capture Microdissection using PixCell IIe LCM workstation (Arcturus). Note absence of target structure (dentate gyrus). (C) Microdissected dentate gyrus adhered to LCM cap. The thermoplastic film with captured cells can be added directly to the RNAqueous-Micro lysis solution from the RNA extraction.

Figure 2. Electropherogram of RNA from Microdissected Dentate Gyrus. RNA was extracted from microdissected tissue using Ambion's RNAqueous-Micro Kit and analyzed on an Agilent 2100 bioanalyzer. RNA concentration =18 ng/l, rRNA [28S/18S rRNA] = 0.89.

Figure 3. Real-time RT-PCR Using RNA From Microdissected Mouse Brain Dentate Gyrus. RT-PCR was performed in duplicate RNA from the microdissected dentate gyrus. The assay was run on an ABI PRISM 7000 Sequence Detection System with primers and probe for Mouse GAPDH. Amplification from left to right represents female 810 wk and male 26 wk dentate gyrus, and minus-RT controls.

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