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Isolation and detection of RNA by co-immunoprecipitation
The discovery of non-coding RNA has reignited the interest in RNA research, making it a central focus in life sciences. Since RNA is inherently unstable, most RNA molecules must interact with specific RNA-binding proteins to form stable RNA/protein complexes within the cell. These interactions are dynamic and play a critical role throughout the entire RNA life cycle—starting from transcription and processing, through transport and localization, to function and degradation. As a result, isolating or identifying RNA-binding proteins is a fundamental technique in RNA research. This process typically involves immunoprecipitating RNA-protein complexes using antibodies against specific RNA-binding proteins, followed by isolation of the associated RNA. The isolated RNA can then be analyzed using end-labeling and denaturing gel electrophoresis, or more recently, through high-throughput RNA sequencing for comprehensive sequence profiling.
All reagents used in the procedure were either DEPC-treated or prepared in DEPC water to prevent RNase contamination. During RNA extraction, the rinsing step can be omitted if necessary. For RNA sample dissolution and storage, TE buffer (pH 8.0) is recommended. If the next step involves enzymatic reactions, the RNA should be stored at 80°C. Alternatively, if electrophoresis is planned, 100% deionized formamide is preferred.
Proper handling of RNA is crucial to avoid degradation. Working in an RNAZap environment helps minimize RNase contamination. The immunoprecipitation of RNP complexes begins with tissue or cell fixation, where UV cross-linking may be applied to stabilize RNA-protein interactions. Cells or tissues are washed twice with cold PBS before being lysed. The lysis buffer contains Tris-HCl, KCl, EDTA, NP40, NaF, DTT, RNasin, and protease inhibitors. After centrifugation, the supernatant is collected for immunoprecipitation. Protein A/G Sepharose beads are pre-equilibrated and incubated with the antibody at 4°C for several hours. The sample is then added, and the mixture is incubated further to allow binding. Following centrifugation, the beads are washed with low- and high-salt buffers to remove non-specific interactions.
To extract small RNA, the beads are resuspended in Proteinase K solution and digested at 55°C for 10 minutes. RNA is then extracted using the TRizol method and precipitated with ethanol. For 5' end labeling, the RNA is treated with Calf Intestinal Phosphatase to remove the 5' phosphate group, followed by phenol-chloroform extraction and ethanol precipitation. The RNA is then labeled with γ-32P ATP and separated on a 15% polyacrylamide urea gel. Autoradiography is used to visualize the results. Finally, high-throughput RNA sequencing, such as Solexa, provides detailed insights into the RNA profile.