Pena John T G, Sohn-Lee Cherin, Rouhanifard Sara H, Ludwig Janos, Hafner Markus, Mihailovic Aleksandra, Lim Cindy, Holoch Daniel, Berninger Philipp, Zavolan Mihaela, Tuschl Thomas
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, New York 10065, USA.
Nat Methods. 2009 Feb;6(2):139-41. doi: 10.1038/nmeth.1294. Epub 2009 Jan 11.
MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.
微小RNA是具有多种生物学功能并与疾病相关的小调控RNA。我们发现，使用传统甲醛固定进行原位杂交（ISH）会导致小鼠组织切片中大量微小RNA丢失，而使用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺固定可防止这种情况，该试剂能将微小RNA不可逆地固定在其5'磷酸基团上。我们通过在ISH过程中记录核酸解链温度，确定了130种锁核酸探针的最佳杂交参数。
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