miRNA in situ hybridization in formaldehyde and EDC-fixed tissues.
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.
Pena
John T G
JT
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, New York 10065, USA.
Sohn-Lee
Cherin
C
Rouhanifard
Sara H
SH
Ludwig
Janos
J
Hafner
Markus
M
Mihailovic
Aleksandra
A
Lim
Cindy
C
Holoch
Daniel
D
Berninger
Philipp
P
Zavolan
Mihaela
M
Tuschl
Thomas
T
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D001923:Brain Chemistry; D002234:Carbodiimides / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*
C518491:1-ethyl-3-(3-(diethylamino)propyl)carbodiimide; D002234:Carbodiimides; D035683:MicroRNAs; D005557:Formaldehyde