Patrick Micke

Biobanking of fresh frozen tissue : RNA is stable in nonfixed surgical specimens

Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6–16 h. Gene expression by real-time PCR of cfos, HIF1α, Bcl2, PCNA, TGFβ1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2–3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.

Dynamic changes in the expression of DEP-1 and other PDGF receptor-antagonizing PTPs during onset and termination of neointima formation

Growth factor‐dependent tissue remodeling, such as restenosis, is believed to be predominantly regulated by changes in expression of receptor‐tyrosine‐kinases (RTKs) and their ligands. As endogenous antagonists of RTKs, protein‐tyrosine‐phosphatases (PTPs) are additional candidate regulators of these processes. Using laser‐capture‐microdissection and quantitative RT‐polymerase chain reaction (qRT‐PCR), we investigated the layer‐specific expression of the four platelet‐derived growth factor (PDGF) isoforms, the PDGF‐ and receptors, and five PTPs implied in control of PDGF‐receptor signaling 8 and 14 days after balloon injury of the rat carotid. Results were correlated with analyses of PDGF‐ receptor phosphorylation and vascular smooth muscle cell (VSMC) proliferation in vivo. The expression levels of all components, as well as receptor activation and VSMC proliferation, showed specific changes, which varied between media and neointima. Interestingly, PTP expression—particularly, DEP‐1 μlevels—appeared to be the dominating factor determining receptor‐phosphorylation and VSMC proliferation. In support of these findings, cultured DEP‐1–/–cells displayed increased PDGF‐dependent cell signaling. Hyperactivation of PDGF‐induced signaling was also observed after siRNA‐down‐regulation of DEP‐1 in VSMCs. The results indicate a previously unrecognized role of PDGF‐receptor‐targeting PTPs in controlling neointima formation. In more general terms, the observations indicate transcriptional regulation of PTPs as an important mechanism for controlling onset and termination of RTK‐dependent tissue remodeling.—FASEB J. 21, 523–534 (2007)

Laser assisted cell microdissection using the PALM system

Laser-assisted microdissection has enabled the collection of morphologically defined cell populations from a tissue section. The PALM Robot MicroBeam laser microdissection system provides a robust system for the retrieval of specified cells (including single cells). Due to the fragile nature of DNA, and in particular RNA, robust protocols are required to obtain reliable data from a limited number of cells (1-10.000 cells). This chapter describes the application of the PALM MicroBeam system to isolate RNA and DNA from cells in a complex tissue for subsequent molecular analysis. Protocols for successful analysis of RNA from 500 to 1000 cells, including steps to produce cDNA for subsequent polymerase chain reaction analysis, are given. The cDNA could also be used as a template for linear amplification in order to perform gene array analysis. Furthermore, a protocol for genomic analysis of p53 mutations from single cells is given. The described procedures emphasize preparation of tissue, laser microdissection including catapulting of cells, and extraction of RNA and DNA. Downstream experiments for validation are also shown.

A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting.

Laser microdissection and pressure catapulting has become a powerful tool to obtain homogeneous cell populations from tissue samples in nearly all fields of biomedical research. The isolated cells can be subsequently used for the analysis of proteins, DNA or RNA. However, the method requires physical access to the tissue surface and the sections therefore need to be air‐dried and uncovered. The consequence is poor morphology, which severely reduces the potential of the technique, especially in non‐homogeneous tissues or tissues with infiltrating immune cells. To overcome this limitation, a fluid cover medium was developed and the effects on frozen and paraffin wax‐embedded tissue morphology were evaluated. The cover medium improved the morphology such that it was almost comparable to sections overlaid with glass coverslips. Moreover, the laser microdissection procedure was facilitated, since the medium allowed larger areas of tissues to be laser pressure‐catapulted. Neither the isolation of proteins nor the extraction of genomic DNA was adversely affected by the use of the fluid cover medium. No significant differences in RNA quantity and integrity were detected by TaqMan real‐time PCR for GAPDH, and microchip electrophoresis, between covered and uncovered tissue sections. In conclusion, this method provides considerably improved morphology for laser microdissection and pressure catapulting techniques without affecting RNA‐dependent downstream applications. This not only facilitates established procedures, but will also extend the application to tissues that require superior morphological resolution. Copyright

Regulation of tyrosine phosphatases in the adventitia during vascular remodelling.

Protein tyrosine phosphatases (PTPs) are regulators of growth factor signalling in vascular remodelling. The aim of this study was to evaluate PTP expression in the context of PDGF-signalling in the adventitia after angioplasty. Utilising a rat carotid artery model, the adventitial layers of injured and non-injured vessels were laser microdissected. The mRNA expression of the PDGF beta-receptor, the ligands PDGF-A/B/C/D and the receptor-antagonising PTPs (DEP-1, TC-PTP, SHP-2, PTP1B) were determined and correlated to vascular morphometrics, proliferation markers and PDGF beta-receptor phosphorylation. The levels of the PDGF beta-receptor, PDGF-C and PDGF-D were upregulated concurrently with the antagonising PTPs DEP-1 and TC-PTP at day 8, and normalised at day 14 after vessel injury. Although the proliferation parameters were time-dependently altered in the adventitial layer, the phosphorylation of the PDGF beta-receptor remained unchanged. The expression dynamics of specific PTPs indicate a regulatory role of PDGF-signalling also in the adventitia during vascular remodelling.