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Factors affecting the yield of microRNAs from laser microdissectates of formalin-fixed tissue sections

ABSTRACT: Sixth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to profile in duplicate the expression of microRNAs in 250 or 400 ng of RNA isolated from a laser microdissectate of a formalin-fixed and paraffin-embedded human non-small cell lung cancer tumor.

ORGANISM(S): Merkel cell polyomavirus Mus musculus Human gammaherpesvirus 8 Rattus norvegicus Human alphaherpesvirus 1 JC polyomavirus Betapolyomavirus macacae Human immunodeficiency virus 1 Murid gammaherpesvirus 4 Homo sapiens Human betaherpesvirus 5 Human alphaherpesvirus 2 Betapolyomavirus hominis human gammaherpesvirus 4 Mus musculus cytomegalovirus 2 Murid betaherpesvirus 1

PROVIDER: GSE31946 | GEO | 2011/09/08

SECONDARY ACCESSION(S): PRJNA145011

REPOSITORIES: GEO

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Factors affecting the yield of microRNAs from laser microdissectates of formalin-fixed tissue sections

Project description:Sixth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to profile in duplicate the expression of microRNAs in 250 or 400 ng of RNA isolated from a laser microdissectate of a formalin-fixed and paraffin-embedded human non-small cell lung cancer tumor. Laser microdissection was performed on a hematoxylin-eosin-stained, 8 um-thick section of formalin-fixed and paraffin-embedded tissue. The microdissectate was treated with proteinase K overnight at 55 ºC. Total RNA from the lysate was then extracted using FFPE RNA Isolation Kit from Norgen Biotek® (Thorold, Canada). RiboGreen assay was used to quantify RNA in the preparation. Two-hundred-fifty or 400 ng of RNA was used in duplicate for dual-channel microarray-based microRNA expression profiling. Sample RNA was labeled with the Cy3-like Hy3™ dye, mixed with a human universal reference RNA (product number AM6000, Ambion®, Austin, TX) labeled with the Cy5-like Hy5™ dye, and hybridized to the two-color miRCURY™ arrays.

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Factors affecting the yield of microRNAs from laser microdissectates of formalin-fixed tissue sections.

Project description:BACKGROUND:Quantification of microRNAs in specific cell populations microdissected from tissues can be used to define their biological roles, and to develop and deploy biomarker assays. In this study, a number of variables were examined for their effect on the yield of microRNAs in samples obtained from formalin-fixed paraffin-embedded tissues by laser microdissection. RESULTS:MicroRNA yield was improved by using cresyl violet instead of hematoxylin-eosin to stain tissue sections in preparation for microdissection, silicon carbide instead of glass fiber as matrix in RNA-binding columns, and overnight digestion of dissected samples with proteinase K. Storage of slides carrying stained tissue sections at room temperature for up to a week before microdissection, and storage of the microdissectates at room temperature for up to a day before RNA extraction did not adversely affect microRNA yield. CONCLUSIONS:These observations should be of value for the efficient isolation of microRNAs from microdissected formalin-fixed tissues with a flexible workflow.

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Factors affecting the yield of microRNAs from laser microdissectates of formalin-fixed tissue sections

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