Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes.
MicroRNAs (miRNAs) constitute a group of small non-coding RNA molecules generally 18-22 base-pairs in length. miRNAs are considered to be negative regulators of gene expression at the level of post-transcription and are important in normal physiological development, tissue homeostasis and disease. The cellular origin of individual microRNAs is of utmost importance for understanding their roles in molecular and biological processes, in multi-cellular and complex structured tissues. For the localization of miRNAs in clinical and experimental formalin-fixed and paraffin-embedded samples we have developed a simple and robust one-day in situ hybridization protocol based on the use of double digoxigenin (DIG)-labeled LNA-DNA chimeric probes. We show that the protocol enables analyses of specificity, and demonstrate the detection of miR-1, miR-21, miR-124, miR-126, miR-145, and miR-205 in human and murine paraffin material. The well established localization of these microRNAs makes them ideal for use as reference microRNAs when optimizing the microRNA in situ hybridization assay for a particular tissue and miRNA.
Jørgensen
Stine
S
Exiqon A/S, Diagnostic Product Development, Skelstedet 16, Vedbæk, Denmark.
Baker
Adam
A
Møller
Søren
S
Nielsen
Boye Schnack
BS
D000818:Animals; D001943:Breast Neoplasms / Q000737:chemistry; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D035683:MicroRNAs / Q000032:analysis*; D009841:Oligonucleotides; D016612:Paraffin Embedding; D012342:RNA, Small Nuclear; D012680:Sensitivity and Specificity
D035683:MicroRNAs; D009841:Oligonucleotides; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; C477371:locked nucleic acid; D019286:Endopeptidase K