In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets.
This manuscript details a protocol for the co-localization of a microRNA and its putative protein target in paraffin embedded formalin fixed tissues. The key variables for the first step, microRNA in situ hybridization, includes probe concentration (1-2 pmol/μl), locked nucleic acid (LNA) modified probes, protease digestion (pepsin 1.3mg/ml), and a low stringency wash. Key variables for the subsequent immunohistochemical step are the concentration of the primary antibody, proper pretreatment (none, proteinase K, or antigen retrieval), and use of a highly sensitive detection system. A computer based system can convert the colorimetric signals (blue chromogen (NBT/BCIP) for the microRNA, and either a red (fast red) or brown (DAB) chromogen for the protein) to distinct fluorescent-based colors, and then mix them to determine if a given cell has the microRNA and protein of interest. Co-expression of a microRNA and its putative target in tissue sections offers physiologic corroboration of solution-based methods that a given microRNA may be regulating a specific protein.
Nuovo
Gerard J
GJ
The Comprehensive Cancer Center, Ohio State University Medical Center, Columbus, OH, USA. gnuovomd@pol.net
D000818:Animals; D005557:Formaldehyde; D017403:In Situ Hybridization / Q000379:methods*; D035683:MicroRNAs / Q000032:analysis* / Q000378:metabolism; D009841:Oligonucleotides; D016612:Paraffin Embedding / Q000379:methods; D010434:Pepsin A / Q000378:metabolism; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D015347:RNA Probes; D012333:RNA, Messenger / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods
D035683:MicroRNAs; D009841:Oligonucleotides; D011506:Proteins; D015347:RNA Probes; D012333:RNA, Messenger; C477371:locked nucleic acid; D005557:Formaldehyde; D010434:Pepsin A