RNAs isolated using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Product No. 17-700) can be analyzed by several molecular methods including end- point RT-PCR and quantitative RT-PCR (if binding targets of the RBP are known), or by microarray or deep sequencing methods. Given RNA targets of known sequence, gene specific primers can be designed that allow validation (and quantification) of the RNA immunoprecipitated by the antibodies used. Once successful RIP can be confirmed, further interrogation of the population of RNAs in an immunoprecipitation may be pursued by population based methods such as comparative microarray hybridization of resulting cDNAs or by deep sequencing of molecularly adapted products of the RIP reaction (see Baroni, T.E. et al., 2008. Methods Mol Biol. 419:93-108).
Presented below are methods for performing end-point or real-time quantitative measurement of RIP experiments using the control antibody supplied in the EZ-Magna RIP™ kit (Product No.17-701), Anti-SNRNP70.
For reverse transcription, use any commercially available reverse transcription enzymes and kit systems that use random hexamers for priming. Because the mature U1 snRNA co-precipitated with U1 spliceosomal SNRNP70 protein is not polyadenylated, oligo d(T) priming is not recommended.
Note: RNase-free aerosol resistant tips are recommended for use in this section to minimize risk of contamination.
Standard end-point RT-PCR
Real-time Quantitative PCR
For more information about RIP experiment guides, troubleshooting tips and supplementary protocols, please view our RNA Immunoprecipitation (RIP) page.