Enhanced molecular analyses by combination of the HOPE-technique and laser microdissection

Abstract

As part of an investigation aimed at illuminating the possibilities and limits of the HOPE-fixation and paraffin-embedding technique we here describe a novel procedure which was developed in order to combine the benefits of the HOPE-technique with the capabilities of laser microdissection. The presented procedure avoids the need for amplification of template-RNA thus facilitating reliable and reproducible results. The excellent preservation of nucleic acids, proteins, and morphology in HOPE-fixed, paraffin-embedded tissues enhances the molecular applications available to date with materials acquired by laser microdissection when compared to formalin fixed, paraffin-embedded tissues, thus substantially extending the methodological panel in tissue based research.

Figure 1

Photomicrographs of HOPE-fixed tissues, deparaffinized and stained with cresyl violet (both 100× magnification). Sections were subjected to LMPC utilizing a PALM MicroBeam system. A: Pulmonary carcinoma with the area subjected for LMPC marked. B: Pulmonary carcinoma with the area subjected for LMPC microdissected and transferred to a microfuge tube by pressure catapulting.

Figure 2

Results of RNA-integrity-testing by a Agilent Bioanalyzer 2100 using the RNA 6000 Pico LabChip kit. Lane 1 and 2 are HOPE-fixed materials and lane 3 is FFPE.

Figure 3

Real time RT-PCR targeting a 231 bp fragment of the RNA of human Hypoxanthine phosphoribosyltransferase. Green: HOPE, deparaffinized with xylene, cut out section. Black drawn through: HOPE, deparaffinized with isopropanol, cut out section. Black with dots: HOPE, isopropanol, whole slice before mounting on slide directly processed. Blue: HOPE, deparaffinized with isopropanol, LMPC. Red: HOPE, deparaffinized with xylene, LMPC. Black noncontinuous: two superposing samples, FFPE, both deparaffinized with xylene, cut out section and LMPC