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Dig RNA Probe Synthesis and Purification
Published: Jan 20, 2012 DOI: 10.21769/BioProtoc.173 Views: 30069
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Abstract
In situ hybridization is an effective method to examine the expression level and location of a gene of interest in tissues or cells. To do this, RNA can be labeled with digoxigenin-UTP (DIG) by in vitro transcription with SP6 and T7 RNA polymerase. The method provided in this protocol is a detailed description of synthesizing an antisense DIG-labeled RNA probe for in situ hybridization using the DIG RNA labeling kit from Roche.
Background
Materials and Reagents
- CI (Chloroform: Isoamylalcohol = 24:1)
- NaOAc
- EtOH
- LiCl
- DEPC H2O
- RNasin Plus Protease Inhibitor (Promega Corporation, catalog number: N2611 )
- 70% ethanol
- PCI (Phenol: Chloroform: Isoamylalcohol = 25:24:1, volume, Sigma-Aldrich, catalog number: P3802 )
- Glycogen (QIAGEN, catalog number: 158930 )
- DIG RNA labeling kit (Roche Diagnostics, catalog number: 11175025910 )
- Quick Spin Columns (Roche Diagnostics, catalog number: 1274015 )
Equipment
- Standard tabletop centrifuges
- RNase-free Eppendorf tube
- NanoDrop
- Falcon snap cap tube
- Water bath
Procedure
Category
Molecular Biology > RNA > RNA synthesis
Molecular Biology > RNA > RNA labeling
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