High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection.
BACKGROUND
Laser capture microdissection enables the isolation of single cells or small cell groups from histological sections under direct microscopic observation. Combined with quantitative PCR or microarray, it is a very powerful approach for studying gene expression profiles in discrete cell populations. The major challenge for such studies is to obtain good quality RNA from small amounts of starting material.


RESULTS
We have developed a simple, flexible, and low-cost method for simultaneously producing RNA from discrete cell groups in embryonic day 15 mouse brain. In particular, we have optimized the following key steps in the procedure: staining, cryosectioning, storage of sections and harvesting of microdissected cells. We obtained the best results when staining 20 mum-thick sections with 1% cresyl violet in 70% ethanol and harvesting the microdissected tissue in RNA stabilization solution. In addition, we introduced three stop-points in the protocol which makes the tedious process of laser capture microdissection more flexible, without compromising RNA quality.


CONCLUSIONS
Using this optimized method, we have consistently obtained RNA of high quality from all four simultaneously microdissected cell groups. RNA integrity numbers were all above 8, and long cDNA fragments (> 1.2 kb) were successfully amplified by reverse transcription PCR from all four samples. We conclude that RNAs isolated by this method are well suited for downstream quantitative PCR or microarray studies.
Wang
Wei-Zhi
WZ
Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, Oxford, UK. wei-zhi.wang@dpag.ox.ac.uk
Oeschger
Franziska M
FM
Lee
Sheena
S
Molnár
Zoltán
Z
D000818:Animals; D001921:Brain / Q000166:cytology / Q000601:surgery*; D001923:Brain Chemistry; D015925:Cryopreservation; D016616:Cryoultramicrotomy; D053685:Laser Therapy; D051379:Mice; D042282:Microdissection / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012313:RNA