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.
Micke
Patrick
P
Cancer Center Karolinska, Department of Oncology and Pathology, Karolinska Institute, Stockholm, Sweden.
Ostman
Arne
A
Lundeberg
Joakim
J
Ponten
Fredrik
F
D000818:Animals; D006801:Humans; D007834:Lasers; D042282:Microdissection / Q000295:instrumentation* / Q000379:methods*; D009369:Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology*; D012371:Robotics