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High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection.

Wang WZ, Oeschger FM, Lee S, Molnár Z - BMC Mol. Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, Oxford, UK. wei-zhi.wang@dpag.ox.ac.uk

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.

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.

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.

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Flow diagram of the optimized protocol with possible stop-points (red boxes). All steps were performed in an RNase -free environment. Fresh-frozen whole heads were stored at -80°C for up to several months. Heads were cryosectioned at 20 μm, and four to six sections were mounted on a membrane-coated PEN slide. Sections were fixed in 95% EtOH, rehydrated with 75% and 70% EtOH, stained with 1% cresyl violet in 70% EtOH and dehydrated with 70%, 75%, 95%, 100%, 100% EtOH, 30 sec each. Slides were stored at -80°C up to several weeks in a box with desiccant beads. Four cell groups from the same section were microdissected, harvested in individual tubes with RNA stabilization solution and stored at -80°C. Stabilization solution was replaced with lysis buffer and cell lysates from four brains were pooled before RNA extraction.
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Figure 5: Flow diagram of the optimized protocol with possible stop-points (red boxes). All steps were performed in an RNase -free environment. Fresh-frozen whole heads were stored at -80°C for up to several months. Heads were cryosectioned at 20 μm, and four to six sections were mounted on a membrane-coated PEN slide. Sections were fixed in 95% EtOH, rehydrated with 75% and 70% EtOH, stained with 1% cresyl violet in 70% EtOH and dehydrated with 70%, 75%, 95%, 100%, 100% EtOH, 30 sec each. Slides were stored at -80°C up to several weeks in a box with desiccant beads. Four cell groups from the same section were microdissected, harvested in individual tubes with RNA stabilization solution and stored at -80°C. Stabilization solution was replaced with lysis buffer and cell lysates from four brains were pooled before RNA extraction.

Mentions: The complete optimized protocol is summarized in Figure 5. Using this protocol, we obtained RNA of excellent quality from all four cell groups and importantly reduced the variability between samples. Quality was assessed using the RIN, which has been reported as the most reliable method for evaluating RNA quality in LCM samples [5]. RIN is based on a software algorithm developed by Agilent Technologies that determines RNA integrity using multiple features of the electropherogram [18]. The four cell groups collected had RINs ranging from 8.0 to 8.9 (Figure 4e–i), indicating that RNA is of particularly high quality for downstream applications [6].


High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection.

Wang WZ, Oeschger FM, Lee S, Molnár Z - BMC Mol. Biol. (2009)

Flow diagram of the optimized protocol with possible stop-points (red boxes). All steps were performed in an RNase -free environment. Fresh-frozen whole heads were stored at -80°C for up to several months. Heads were cryosectioned at 20 μm, and four to six sections were mounted on a membrane-coated PEN slide. Sections were fixed in 95% EtOH, rehydrated with 75% and 70% EtOH, stained with 1% cresyl violet in 70% EtOH and dehydrated with 70%, 75%, 95%, 100%, 100% EtOH, 30 sec each. Slides were stored at -80°C up to several weeks in a box with desiccant beads. Four cell groups from the same section were microdissected, harvested in individual tubes with RNA stabilization solution and stored at -80°C. Stabilization solution was replaced with lysis buffer and cell lysates from four brains were pooled before RNA extraction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713242&req=5

Figure 5: Flow diagram of the optimized protocol with possible stop-points (red boxes). All steps were performed in an RNase -free environment. Fresh-frozen whole heads were stored at -80°C for up to several months. Heads were cryosectioned at 20 μm, and four to six sections were mounted on a membrane-coated PEN slide. Sections were fixed in 95% EtOH, rehydrated with 75% and 70% EtOH, stained with 1% cresyl violet in 70% EtOH and dehydrated with 70%, 75%, 95%, 100%, 100% EtOH, 30 sec each. Slides were stored at -80°C up to several weeks in a box with desiccant beads. Four cell groups from the same section were microdissected, harvested in individual tubes with RNA stabilization solution and stored at -80°C. Stabilization solution was replaced with lysis buffer and cell lysates from four brains were pooled before RNA extraction.
Mentions: The complete optimized protocol is summarized in Figure 5. Using this protocol, we obtained RNA of excellent quality from all four cell groups and importantly reduced the variability between samples. Quality was assessed using the RIN, which has been reported as the most reliable method for evaluating RNA quality in LCM samples [5]. RIN is based on a software algorithm developed by Agilent Technologies that determines RNA integrity using multiple features of the electropherogram [18]. The four cell groups collected had RINs ranging from 8.0 to 8.9 (Figure 4e–i), indicating that RNA is of particularly high quality for downstream applications [6].

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, Oxford, UK. wei-zhi.wang@dpag.ox.ac.uk

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.

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.

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.

Show MeSH
Related in: MedlinePlus