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.
Micke
Patrick
P
Ludwig Institute for Cancer Research, Uppsala, Sweden. patrick.micke@licr.uu.se
Bjørnsen
Tone
T
Scheidl
Stefan
S
Strömberg
Sara
S
Demoulin
Jean-Baptiste
JB
Ponten
Fredrik
F
Ostman
Arne
A
Lindahl
Per
P
Busch
Christer
C
D002280:Basal Cell Carcinoma / Q000473:pathology; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016591:Histocytological Preparation Techniques / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007834:Lasers; D008168:Lung / Q000473:pathology; D042282:Microdissection / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016612:Paraffin Embedding / Q000379:methods; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D004273:DNA, Neoplasm; D011506:Proteins; D012334:RNA, Neoplasm; D012313:RNA; D004247:DNA