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High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection.
- Source :
-
BMC molecular biology [BMC Mol Biol] 2009 Jul 06; Vol. 10, pp. 69. Date of Electronic Publication: 2009 Jul 06. - Publication Year :
- 2009
-
Abstract
- 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.<br />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.<br />Conclusion: 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.
Details
- Language :
- English
- ISSN :
- 1471-2199
- Volume :
- 10
- Database :
- MEDLINE
- Journal :
- BMC molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 19580671
- Full Text :
- https://doi.org/10.1186/1471-2199-10-69