Molecular Interaction Experiment: RIP (RNA Immunoprecipitation)

1、 Technical Introduction

RIP (RNA Immunoprecipitation) technology is a key experimental method for studying the interaction between RNA and protein in cells, which can reveal the functional mechanism of RNA binding protein (RBP) in post transcriptional regulation.

2、 Technical principles

RIP uses antibodies targeting the target protein to precipitate the corresponding RNA protein complex, and through separation and purification, performs RT-PCR validation or sequencing analysis on the RNA bound to the complex, resulting in more biologically convincing results.

3、 Application scenarios

1. Basic mechanism: Analyze the function of RNA binding proteins (such as AGO2, Polycomb complex) and RNA regulatory network.

2. Disease research: Compare the differences in RNA protein interactions between diseases and normal samples, and identify potential biomarkers (such as cancer-related lncRNAs).

3. Drug development: Evaluate the impact of drugs on RNA protein interactions to accelerate target screening.

4、 Technical advantages

1. High sensitivity: can detect weak interactions between low abundance RNA and proteins.

2. Wide dynamic range: applicable to a small number of cells (such as 5 × 10 ⁷ cells) to complex tissue samples.

3. Multi group collaboration: Combining RNA Seq, CLIP Seq, or ChIP Seq to construct a multidimensional regulatory network.

5、 Sample type

Fresh cells (adherent cells/suspended cells), frozen cells, frozen tissues, and cross-linked cell samples.

6、 Sample requirements

Cell samples: approximately 1 × 10 ^ 7 (can be stored in a 10cm dish, suspended in a centrifuge tube, or in a T75 bottle);

Frozen tissue: approximately 70mg; cross-linked cell sample: cells treated with 1% formaldehyde cross-linking.

7、 Sample preparation&transportation

Live cell samples: Fresh cell samples stored in dishes/T75 bottles/centrifuge tubes can be transported by express delivery at room temperature (with the latest delivery the next day) for those who are closer. Within the city, they can be delivered by flash or in person. For those who are farther away, cells can be frozen at -80 ℃ and transported by dry ice;

Organizational samples&cross-linked cell samples: frozen at -80 ℃ and transported in dry ice.

8、 Required reagents

Detect ChIP grade antibodies.

9、 Experimental cycle

1-2 weeks (excluding sequencing)

10、 Other

When selecting RT PCR for subsequent testing, the sites for subsequent testing must be provided.

Specific steps for formaldehyde crosslinking cells: ① Add 1% formaldehyde to the cell dish and incubate at 37 ℃ for 10 minutes; ② Wash the cells twice with cold DPBS containing [1mM PMSF, 1 μ g/ml aprotinin, and 1 μ g/ml pepstatin A]. ③ Scrape the cells into 2ml of cold DPBS containing protease inhibitors, transfer to centrifuge tubes, and freeze for shipment.