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

Molecular differences between cortical and trabecular bone, of relevance to understand the pathophysiological bases of bone diseases, can be determined only throughout effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, combining 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-to-280 nm and 260-to-230 nm absorbance- ratios, and 28S-to-18S rRNA ratio. Protein integrity and quality were evaluated by Comassie Blue staining. RT-qPCR and immunoblotting for bone-specific genes and proteins were performed to verify the suitability of the isolated material in downstream applications. 260-to-280 nm and 260-to-230 nm absorbance ratios were, on average, ≥1.8. Bands on agarose gel were consistent to an intact RNA, with mean 28S-to-18S ratios of1.68±0.35 and 1.88±0.10 for cortical and trabecular bone, respectively. Band patterns after Comassie 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 here presented is appropriate and effective for the study of human bone.若要阐明骨骼疾病的病理生理学基础、解析皮质骨（cortical bone）与骨小梁骨（trabecular bone）之间的分子差异，唯有通过高效的RNA与蛋白质分离方法方可实现。本研究提出一种基于TRIzol试剂的方法，通过结合骨骼粉碎与匀浆步骤，可从同一供体的人类皮质骨与骨小梁骨中同时提取总RNA与蛋白质。通过260 nm与280 nm、260 nm与230 nm的吸光度比值，以及28S rRNA与18S rRNA的比值，对RNA的完整性与纯度进行评估；采用考马斯亮蓝染色法对蛋白质的完整性与质量进行评价。针对骨特异性基因与蛋白质开展实时定量聚合酶链反应（RT-qPCR）与免疫印迹（immunoblotting）实验，以验证分离得到的样本是否适用于下游实验。260 nm/280 nm与260 nm/230 nm吸光度比值的平均值均≥1.8。琼脂糖凝胶电泳条带与完整RNA的特征相符，皮质骨与骨小梁骨的28S rRNA/18S rRNA比值平均值分别为1.68±0.35与1.88±0.10。考马斯亮蓝染色后的条带图谱证实了蛋白质的完整性。成功实现了基因与蛋白质表达分析，且两种骨组分间呈现出具有生物学意义的表达差异，进一步验证了该分离样本适用于下游实验。本研究所提出的方法适用于且可有效应用于人类骨骼的相关研究。