Quantitative Molecular Analysis of Laser-Microdissected Paraffin- Embedded Human Tissues

Abstract

Laser microdissection enables the contamination-free isolation of morphologically defined pure cell populations from archival formalin-fixed paraffin-embedded tissue specimens. Cells isolated by this method have been characterized by a wide variety of qualitative molecular assays, e.g. loss of heterozygosity, point mutations, clonality and lineage origin. The recently introduced real-time PCR technology renders the reliable quantification of very small amounts of nucleic acids possible. Several groups including our own showed that this technique can be successfully applied for the quantification of DNA and RNA isolated from microdissected archival tissue sections, even after immunohistochemical staining. The exact analysis of quantitative changes of nucleic acids during the course of pathological alterations has thus become possible. In many situations these quantitative changes can be expected to be more important than qualitative changes. The new technology for the quantification of structural genomic alterations and changes in the gene expression pattern in conjunction with microdissection have equipped morphologists with a powerful tool to study reactive and neoplastic changes of tissues.

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

“…Laser microdissection is best carried out using archival formalin-fixed paraffin-embedded tissue specimens (Lehmann et al 2000) as such tissue is the most widely available material for retrospective clinical studies and, together with clinical data, represents an important resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. Although RNA can be extracted from such tissue (Rupp & Locker 1988), extensive degradation can occur before (Mizuno et al 1998) or during (Klimecki et al 1994) the formalin fixation process.…”

Megakaryocytes from chronic myeloproliferative disorders show enhanced nuclear bFGF expression

“…Total RNA was extracted and reversely transcribed 6 from 50 to 100 megakaryocytes per case isolated by laser-microdissection (P.A.L.M., Wolfratshausen, Germany) from tissue sections of bone marrow. 7 After linearity of PCR amplification over a broad concentration range and equal efficiencies for all primers/probe systems could be shown, relative quantification of bFGF and ␤-glucuronidase (␤-GUS) mRNA was performed in 2 independent runs using the ⌬⌬CT-method. 8 Megakaryocytes from PV, ET, and prefibrotic and fibrotic IMF displayed exaggerated bFGF mRNA levels compared to megakaryocytes from normal or reactive controls and CML (P Ͻ .0001, respectively).…”

Quantitative Gene Expression Profiling in Formalin-Fixed, Paraffin-Embedded Tissues Using Universal Bead Arrays

“…10 As a result, most gene expression analysis of FFPE tissues has so far been done using immunohistochemical staining (IHC) and quantitative RT-PCR (qPCR), which allow only a few genes to be analyzed at a time. 9,[11][12][13][14][15][16] Although sufficient RNA can be isolated from a few 10-m slide-mounted paraffin sections to quantitate up to 30 genes by qPCR, 17 there is clearly a bottleneck in scaling up the number of genes that can be measured by this approach. Also, qPCR does not reliably measure RNA fragments shorter than 100 bp.…”