RNA Immunoprecipitation qPCR (RIP- qPCR) Protocol

RNA Immunoprecipitation (RIP)-qPCR

RNAs isolated using the Magna RIP kit 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 illustrative methods for performing end-point or real time quantitative measurement of RIP experiments using the control antibody supplied in the EZ-Magna RIP kit ((17-701), Anti-SNRNP70 (CS203216)).

For reverse transcription, the user for EZ-Magna RIP kit can use any commercially available reverse transcription enzymes and kit systems that use random hexamers for priming. Since the mature U1 snRNA co-precipitated with U1 spliceosomal SNRNP70 protein is not polyadenylated, oligo d(T) priming is not recommended.

Example of 1st strand cDNA synthesis (e.g. High Capacity RNA-to-cDNA Kit, ABI (4387406))

Note: RNase-free aerosol resistant tips are recommended for use in this section to minimize risk of contamination.

1. Label the appropriate number of 0.2 mL PCR tubes for the number of samples to be analyzed and place on ice.
2. Add 9 μL (up to 2 μg of RNA) of the appropriate sample to the PCR tube and return on ice.
3. Add the appropriate amount of reagents to each PCR reaction tube on ice as indicated in Table below.
   

Reagent	Volume for 1 reaction (μL)
RNA	9.0
2X RT Buffer	10.0
20X Enzyme Mix	1.0
Total per reaction	20.0

4. Place the PCR reaction tubes in a thermal cycler.
5. Start the following RT reaction program:
   

RT Reaction	37 °C	60 min
Stop the reaction	95 °C	5 min
Hold	4 °C	Hold

6. Remove the PCR tubes. Dilute the reaction with 180 μL of Nuclease-free water (10X dilution). Reactions can be stored at -20 °C.

Standard end-point RT-PCR

1. Label the appropriate number of 0.2 mL PCR tubes for the number of samples to be analyzed and place on ice.
   • At a minimum, there will be 4 samples to undergo PCR using the RIP Primers included in this kit: cDNA from positive and negative control antibody immunoprecipitations, Input and a no template tube as a control for DNA contamination.
   • The RIP Primers are specific for the human U1 snRNA gene. It is recommended that the user design appropriate specific primers for cDNA from other species and determine the PCR reaction conditions empirically.
2. Add 2 μL of the appropriate sample to the PCR tube and return to ice.
3. Add the appropriate amount of reagents to each PCR reaction tube on ice, adding the H2O first and the Taq polymerase last.
   • It is recommended that the user employ a Hot-Start Taq polymerase. If user is not employing a Hot-Start Taq polymerase, Taq must be added to each tube after the initial denaturation step.
   • If a master reaction mix is desired, dispense enough reagents for one extra tube to account for loss of volume.
4. Place the PCR reaction tubes in a thermal cycler.
5. Start the following PCR reaction program:
   
6. Remove the PCR tubes. Reactions can be stored at -20 °C.
7. Remove 10 μL of each PCR reaction for analysis by 2% agarose gel electrophoresis with a 100 bp DNA marker. For U1 snRNA included in Cat. # 17-701, the expected size of the PCR product is 100 base pairs. See Figure A. (pg. 15) for an example.

Real-time Quantitative PCR

1. Add 2 μL of the cDNA sample to the PCR plate suitable for your real time instrument of choice (Performing a triplicate of qPCR reactions per RIP sample is recommended).
2. Prepare a master reaction mix. Dispense enough reagents for one extra tube to account for loss of volume.
3. Add 23 μL of qPCR mix to the 2 μL of the sample.
4. Use caps or an optical tape to seal the plate and start the qPCR reactions.

For more information about RIP experiment guides, troubleshooting tips and supplementary protocols, please view our RNA Immunoprecipitation (RIP) page.