Protecting rna in fixed tissue (2003)
- Authors:
- USP affiliated authors: MORISCOT, ANSELMO SIGARI - ICB ; BRITTO, DANIA EMI HAMASSAKI - ICB
- Unidade: ICB
- Assunto: HISTOLOGIA
- Language: Português
- Abstract: Objetivo: RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection, in situ hybridization and PCR in situ. In this study, our aim was to test an alternative protocol for paraformaldehyde fixed retinal sections in order to increase RNA yield and improve its quality without impairment in tissue morphology. Métodos e Resultados: Adult mice were sacrificed, eyes were dissected out and retinas were submitted to two different protocols. The tissue was incubated in RNAlater for 1 h (Test group only); fixed in 2% paraformaldehyde in 0.1 M phosphate buffer (PB) for 15 minutes, cryoprotected in 30% sucrose in PB for 2 hours and embedded in OCT. Retinal sections were collected in cell culture dish for immediate RNA isolation using TRIzolÒ, according to instructions provided by the manufacturer. Spectrophotometer analysis indicated that incubation in RNAlater prior to fixation protocol improves RNA yield in about 80% (P<0.01); a slightly improvement in RNA quality was also found (P=0.02). RT-PCR amplification followed by ethidium bromide detection indicated that RNAlater treatment did result in stronger bands for both Cx32 (386-bp) and GAPDH (200-bp) amplicons.) The morphology of frozen OCT-embedded retinal sections showed no significant differences by using the RNA preservative. In addition, immunoreactivity for calbindin, a calcium binding protein,was very similar in retinas of both groups. Conclusões: In conclusion, the use of RNA preservative prior to fixation steps constitutes a useful tip in order to prepare tissues for laser microdissection. It clearly increases RNA yield and improves its quality, with no difference in any histological criteria. Based in our results, this simple modification in protocol could also be useful for users of other techniques, which require both RNA integrity and preserved morphology, such as in situ hybridization and PCR in situ
- Imprenta:
- Publisher: Federação de Sociedades de Biologia Experimental
- Publisher place: Curitiba, Paraná
- Date published: 2003
- Source:
- Título do periódico: Resumos
- Conference titles: Reunião Anual da Federação de Sociedades de Biologia Experimental, FeSBE
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ABNT
KIHARA, A. H. et al. Protecting rna in fixed tissue. 2003, Anais.. Curitiba, Paraná: Federação de Sociedades de Biologia Experimental, 2003. . Acesso em: 30 abr. 2024. -
APA
Kihara, A. H., Moriscot, A. S., Ferreira, P. J., & Hamassaki, D. E. (2003). Protecting rna in fixed tissue. In Resumos. Curitiba, Paraná: Federação de Sociedades de Biologia Experimental. -
NLM
Kihara AH, Moriscot AS, Ferreira PJ, Hamassaki DE. Protecting rna in fixed tissue. Resumos. 2003 ;[citado 2024 abr. 30 ] -
Vancouver
Kihara AH, Moriscot AS, Ferreira PJ, Hamassaki DE. Protecting rna in fixed tissue. Resumos. 2003 ;[citado 2024 abr. 30 ] - Ontogenetic expression of connexins in mouse retina
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- Connexin 31.1 is expressed in mouse retina and modulated by dark adaptation
- O hormônio tireoideano reprime a expressão gênica de proteínas estruturais do disco M no músculo esquelético de ratos
- O hormônio tireoideano reprime a expressão da proteína M no coração de ratos.
- Connexin 31.1 is expressed in mouse retina and modulated by dark adaptation
- RT-PCR for connexin 32 from paraformaldehyde-fixed mouse retina
- RT-PCR for connexins from paraformaldehyde-fixed mouse retina
- O hormônio tireoideano reprime a expressão da Miomesina
- Thyroid hormone down regulates structural M band proteins in rat heart
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