Robotic MicroRNA In-Situ Hybridization by Locked Nucleic Acid Probes with Advanced Controls
Murat B Yaylaoglu, Scot D Liu, Bronislawa Petryniak, Adrian M Jubb, Nianfeng Ge, Gregor Eichele, Hartmut Koeppen. Genentech Inc., South San Francisco, CA; Max Planck Institute of Biophysical Chemistry, Goettingen, Germany
Background: Resolving the spatial distribution of transcripts at the cellular level is essential to understanding the biology of microRNAs, and may assist in validating their function. However, the majority of in situ hybridization protocols for microRNAs are unable to demonstrate an adequate signal to background ratio, are not high throughput, do not use appropriate controls and cannot prevent hybridization of the probe to partially homologous off target sequences.
Design: This protocol uses locked nucleic acid (LNA) probes on a robotic platform to evaluate microRNA expression patterns via non-radioactive in situ hybridization (ISH) with two enzymatic amplification steps (catalyzed reporter deposition) to vastly increase sensitivity. In addition, this protocol introduces two novel controls.
The first control is an LNA probe binding oligonucleotide, which abolishes the expression signal by trapping the LNA probe, the sequence of which is identical to that of the microRNA to be examined, termed “LTO” (LNA trap oligonucleotide). The second control comprises oligonucleotides that bind to and mask the LNA probe target sequence (that is complementary in sequence to the microRNA to be examined, termed (c)soRNA) and/or partially homologous off-target sequences, a new type of control in the form of synthetic oligomeric RNA (soRNA).
Results: We hybridized sagittal tissue sections from adult mouse brain with a LNA probe. As increasing concentrations of LTO were added the expression signal gradually decreased until it was no longer detectable.
Prehybridizing sagittal sections of adult mouse brain with increasing concentrations of csoRNA designed against the target microRNA, prevented hybridization of the LNA probe to the target.
In the presence of the cocktail, increasing csoRNA concentrations gradually decreased the specific expression signal, which was completely abolished.
Conclusions: These two controls allow us to evaluate the specificity of the LNA probe and determine whether off-target sequences exist and need to be masked, thereby increasing specificity. Proof-of-concept data are presented for mmu-miR-124 expression in mouse brain.
Tuesday, March 20, 2012 1:45 PM
Platform Session: Section E, Tuesday Afternoon