( ...)Laser microdissection and nucleic acid purification - a Leica - QIAGEN ,,, cooperation

Stefanie Schrer, Werner Wittke,* and Christian Kupfer
QIAGEN GmbH, Hilden, Germany

* Leica Microsystems, Wetzlar, Germany Laser microdissection (LMD) provides a powerful technology to isolate specific cells or cell groups for further analysis. This eliminates the problems of cell-type heterogeneity within tissue samples by providing homogeneous cell populations for analysis. The technique allows highly specific comparisons, for example, of cancer cells or other diseased cells with normal cells in the same tissue sample.

Now QIAGEN has joined forces with Leica Microsystems, a leading supplier of highprecision optical microscopes, to bring you complete systems for LMD with nucleic acid purification and analysis of the dissected samples.

Laser Microdissection Using the Leica
AS LMD System A major benefit of laser microdissection using the Leica AS LMD System is excision of cells from sections with the highest precision and without physical contact. Following cutting by a laser, the excised part falls into a collection tube under gravity, ensuring contaminationfree processing and minimizing sample damage. The Leica AS LMD System enables excision of cells or metaphase chromosomes from a wide range of samples, including paraffin-embedded sections, frozen sections,blood smears, and stained and immunolabeled specimens. The Leica Laser Microdissection Microscope provides an easy-to-use, automated method for microdissection (Figure 1).
QIAGEN has developed a set of protocols for isolation and analysis of high-quality DNA and RNA from laser-microdissected samples. The protocols are designed for use with either cryosectioned or formalin-fixed tissues.
Formalin, which is commonly used to fix tissue samples, can cause significant degradation of RNA and DNA in the fixed samples, which affects purification and downstream analysis. Depending on the fixation protocol, the age of the samples, the staining procedure, and the storage conditions used, RNA and DNA may be highly fragmented into pieces smaller than 300 nucleotides, thus limiting the size of fragments isolated. DNA quality from formalinfixed tissues can be significantly improved by cleanup with the QIAquick PCR Purification Kit, as described in the optimized protocol (Figure 2). Flash-frozen, cryosectioned tissues provide higher-quality nucleic acids. For RNA purification and analysis, optimized protocols using the RNeasy Mini Kit are available from QIAGEN. As with DNA isolation, flashfrozen, cryosectioned tissues are preferable
for isolation of RNA. Since the RNeasy procedure removes RNA smaller than 200 nucleotides, this can lead to an overall loss in yield if the RNA is highly degraded. TaqMan analysis demonstrates the better quality of RNA from flash-frozen tissues, as indicated by earlier detection and a lower threshold cycle (Figure 3A and 3B).
Figure 1 Sections of tissue, before (top panel) and after (middle panel) laser microdissection using the Leica AS LMD System, and image of dissected material in the collection tube using inspection mode (bottom panel). DNA from LMD Samples Using Optimized QIAGEN Protocols Figure 2 Cells were laser-microdissected from flash-frozen and formalin-fixed human colon tissue using the Leica AS LMD System. DNA was isolated following the optimized QIAGEN protocol, either with (+) or without () cleanup using the QIAquick PCR Purification Kit. PCR of the GAPDH gene was carried out for 35 cycles using the HotStarTaq Master Mix Kit and primers from QIAGEN Operon. M: markers. TaqMan Analysis of RNA from LMD Samples Figure 3 Cells were laser-microdissected from flash-frozen and formalin-fixed human colon tissue using the Leica AS LMD System. RNA was isolated following the optimized QIAGEN protocols (Protocol 1: optimized protocol for frozen tissue; Protocol 2: optimized protocol for formalin-fixed tissue). Real-time, quantitative RT-PCR was carried out on an ABI PRISM 7700 Sequence Detector using the QuantiTect Probe RT-PCR Kit and primers and probes for -actin or TNF-alpha, as indicated. All reactions were run in quadruplicate. A Sample amplification plot for -actin using RNA purified from flash-frozen tissue. B Threshold cycles for -actin. C Threshold cycles for TNF-alpha. Protocol 2, optimized for formalin-fixed tissues, includes digestion with QIAGEN Proteinase K for higher RNA yield and quality. Using this optimized protocol, transcripts from formalin-fixed tissues were detected one cycle earlier than when using the protocol for cryosections (Figure 3B). Low-abundance transcripts, such as TNF-alpha, can be detected and quantified from lasermicrodissected samples, provided that the samples are properly handled. Using the optimized protocols for each type of lasermicrodissected tissue, TNF- mRNA was easily detected in cryosections but was not detected after 40 cycles with RNA isolated from formalin-fixed tissues (Figure 3C).

Conclusions

• These results emphasize the importance of sample preparation and nucleic acid isolation and analysis from lasermicrodissected samples. Flash-frozen,
  cryosectioned tissues provide a better source for nucleic acid isolation than formalin-fixed tissues.
  
  
• QIAGEN protocols for RNA and DNA isolation have been developed in conjunction with the Leica AS LMD System.

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