Molecular differences between cortical and trabecular bone, of relevance to understanding the pathophysiological basis of bone diseases, can be determined only through effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, which combines bone pulverization and homogenization to extract simultaneously total RNA and proteins from human cortical and trabecular bone from the same carrot. RNA integrity and purity were determined as the 260/280 nm and 260/230 nm absorbance ratios and the 28S/18S rRNA ratio. Protein integrity and quality were evaluated by Coomassie blue staining. Reverse transcription quantitative polymerase chain reaction and immunoblotting for bone-specific genes and proteins were performed to verify the suitability of the isolated material in downstream applications. The 260/280 nm and 260/230 nm absorbance ratios were, on average, less than or equal to 1.8. Bands on agarose gel were consistent with intact RNA, with mean 28S/18S ratios of 1.68 +/- 0.35 and 1.88 +/- 0.10 for cortical and trabecular bone, respectively. Band patterns after Coomassie blue staining confirmed protein integrity. Successful gene and protein expression analysis, with relevant differences between the two compartments, highlighted the suitability of the material in downstream applications. The method presented here is appropriate and effective for the study of human bone.

A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone

Conte, Caterina;Banfi, Giuseppe;
2022

Abstract

Molecular differences between cortical and trabecular bone, of relevance to understanding the pathophysiological basis of bone diseases, can be determined only through effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, which combines bone pulverization and homogenization to extract simultaneously total RNA and proteins from human cortical and trabecular bone from the same carrot. RNA integrity and purity were determined as the 260/280 nm and 260/230 nm absorbance ratios and the 28S/18S rRNA ratio. Protein integrity and quality were evaluated by Coomassie blue staining. Reverse transcription quantitative polymerase chain reaction and immunoblotting for bone-specific genes and proteins were performed to verify the suitability of the isolated material in downstream applications. The 260/280 nm and 260/230 nm absorbance ratios were, on average, less than or equal to 1.8. Bands on agarose gel were consistent with intact RNA, with mean 28S/18S ratios of 1.68 +/- 0.35 and 1.88 +/- 0.10 for cortical and trabecular bone, respectively. Band patterns after Coomassie blue staining confirmed protein integrity. Successful gene and protein expression analysis, with relevant differences between the two compartments, highlighted the suitability of the material in downstream applications. The method presented here is appropriate and effective for the study of human bone.
RNA and protein integrity and purity
RNA isolation
human cortical and trabecular bone
protein and gene expression
protein extraction
Humans
Cancellous Bone
RNA
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/132174
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