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A Novel Sample Preparation Method That Enables Nucleic Acid Analysis from Ultrathin Sections

Published online by Cambridge University Press:  21 March 2013

Vincent P. Klink ,
Giselle Thibaudeau  and
Ronald Altig
Vincent P. Klink*
Affiliation:
Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762, USA
Giselle Thibaudeau
Affiliation:
Institute for Imaging & Analytical Technologies, Mississippi State University, Mississippi State, MS 39762, USA
Ronald Altig
Affiliation:
Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762, USA
*
*Corresponding author. E-mail: vklink@biology.msstate.edu
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Abstract

The ability to isolate and perform nucleic acid analyses of individual cells is critical to studying the development of various cell types and structures. We present a novel biological sample preparation method developed for laser capture microdissection-assisted nucleic acid analysis of ultrathin cell/tissue sections. We used cells of the mitotic bed of the tadpole teeth of Lithobates sphenocephalus (Southern Leopard Frog). Cells from the mitotic beds at the base of the developing teeth series were isolated and embedded in the methacrylate resin, Technovit® 9100®. Intact cells of the mitotic beds were thin sectioned and examined by bright-field and transmission electron microscopy. The cytological and ultrastructural anatomy of the immature and progressively more mature tooth primordia appeared well preserved and intact. A developmental series of tooth primordia were isolated by laser capture microdissection (LCM). Processing of these cells for RNA showed that intact RNA could be isolated. The study demonstrates that Technovit® 9100® can be used as an embedding medium for extremely small tissues and from individual cells, a prerequisite step to LCM and nucleic acid analyses. A relatively small amount of sample material was needed for the analysis, which makes this technique ideal for cell-specific analyses when the desired cells are limited in quantity.

Keywords

plastic embeddinglaser capture microdissectionIllumina®deep sequencingmitotic bedtadpoleteethLithobates sphenocephalusSouthern leopard frogRNA-seq
Type
Equipment and Techniques Development: Biological
Information
Microscopy and Microanalysis , Volume 19 , Issue 3 , June 2013 , pp. 635 - 641
Copyright
Copyright © Microscopy Society of America 2013 

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