Protocol - Magnetic labeling and isolation of biotinylated molecules
A fishing/competition approach used to validate the interaction between lncRNAs and microRNAs by using biotinylated Peptide Nucleic Acid (PNA) oligomers as probes.
Category:
Molecular Biology
Creation date: Oct 10, 2015
Last revision: Jul 15, 2016
Author(s):
Sara Terreri
Contact
Name: Amelia Cimmino
Address: amelia.cimmino@igb.cnr.it
Email: amelia.cimmino@igb.cnr.it
Figure legend:
Expression of miR-596 in J82 cell extracts after retrieval of endogenous uc.8+ with a peptide nucleic acid (PNA)/uc.8+ probe. Data are expressed as the means ± standard deviation (SD) of triplicate values. P values were obtained using the Student t test for independent samples. ***P<0.001 (from Olivieri et al., 2016)
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Total RNA (300 μg) extraction
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RNA incubation with 5'-biotinylated PNA oligomer
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4°C
|
o/n
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with rotation
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μMACS Streptavidin MicroBeads (100 μL, Miltenyi Biotec) addition
|
4°C
|
30'
|
|
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μMACS column equilibration
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100 μL of equilibration buffer for nucleic acids
|
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RNA::PNA complex addition on the top of the μMACS column pre-equilibrated
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Column washes
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4x100 μL washing buffer
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RNA labeled with biotynilated PNA oligomer elution
|
150 μL elution buffer
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Other informations
100 μL μMACS™ Streptavidin MicroBeads bind up to 100 pool biotinylated molecules.
Quality validation:
Yes
Validation info
The protocol has been published in a peer reviewed journal.
Funded by: AIRC
