A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.
As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.
Due to the nature of this experiment, optimization and experience will be the only guaranteed ways to successfully acquire the intended results. In many steps of this procedure, temperature and efficient handling of the reagents and products are critically important. Proper planning and execution of technique will help insure that the experiment was performed in an appropriate timeframe at the optimal temperatures recommended. A major issue with RNA isolation experiments is the sensitivity of RNAs to degradation by RNases. All reagents need to be RNase free and stored or used in RNase-free containers. This is a critical step in ensuring the integrity of your mRNA sample. Even when the experiment is performed properly, however, the desired outcome may not be achieved due to the nature of the interaction between the RBP and its target mRNAs.
One potential problem is having low or even no signal from the RNA isolated by RIP-Chip. Although there may be signal from total RNA, this may be the result of inadequate binding protein being pulled down by the beads. The first troubleshooting step is to confirm that the cellular lysate being used has adequate expression of the specific RBP. Upon confirmation, protein may be isolated after the final NT2 wash and resuspended in Laemmli buffer or another appropriate denaturing buffer and heated at 95 °C for 5 min. Western blot analysis may be used on these samples in coordination with input lysate as well as negative controls to ensure sufficient pull down of associated protein.
Furthermore, because lysing the cell is required to access these components, the potential for abnormal and unwanted interactions between normally separated proteins and mRNA may be introduced. These interactions could potentially bind and “soak up” your target mRNAs or binding proteins through nonspecific interactions. Additionally, proteins in these varying conditions can fold in multiple variations and their binding motifs may become inaccessible to their target mRNAs, preventing their interactions. Both of these strengthen the importance of working efficiently as well as utilizing the optimal temperatures listed to limit these unwanted interactions. Additionally, optimization of washing conditions for each specific target protein will be critical to maximize the purity of the interaction. More stringent washing conditions may be needed. For example, the wash buffer may be supplemented with SDS or an appropriate amount of urea to reduce nonspecific interactions and background in your signal output. This will be completely dependent on the experimenter’s target RBP as well as the target mRNA in their unique physiological conditions. Some conditions will not be suitable for certain mRNA analysis tools, which should be noted in preparation of samples.
Finally, though RIP is successful in the enrichment of RNA-RBP interactions, a well known issue with RIP (CHIP) method is the inability to identify the specific binding domains of the RBP on the transient mRNA targets. Several cross-linking techniques can be used followed by RIP to isolate unique sequence targets; however, the use of short-wave UV tends to lead to nucleic acid damage. A new method known as PAR-CLIP, or photoactivatable ribonucleoside crosslinking and immuoprecipitation, employs long-wave UV to incorporate thiouridine into nascent RNA allowing the identification of unique binding sites from both stable and transient RNA interactions.
Overall, RIP-Chip has been established as an excellent tool used to isolate and study the interactions between RNA binding proteins and their mRNA targets by our group as well as many other research groups. Though sensitive in nature and practice, proper execution of this procedure will yield the isolation of these RNP complexes, which until recently, have been inaccessible for discovery and analysis.
The authors have nothing to disclose.
Department of Defense (Idea Award W81XWH-07-0406) – To Ulus Atasoy
NIH RO1 A1080870 – To Ulus Atasoy
NIH R21 A1079341 – To Ulus Atasoy
University of Missouri Institutional Funds – To Ulus Atasoy
Name of Reagent | Company | Catalog Number | Comments |
1 M dithiothreitol | Fisher | BP172-5 | DTT |
DNase 1 | Ambion | 2235 | RNase free |
Ethylendiamine Tetraccetic Acid | Fisher | BP118-500 | EDTA |
Glycogen | Ambion | 9516 | |
1 HEPES | Sigma | H3375-100G | pH 7.0 |
Igepal Nonidet P-40 | USB | 78641 | NP40 |
1 M KCl | Fisher | BP366-500 | |
1 M MgCl2 | Fisher | BP214-500 | |
NT2 Buffer | *See Below | ||
Polysome Lysis Buffer | *See Below | ||
Protease inhibitor cocktail tablets | Roche | 11873580001 | |
Protein A Sepharose Beads | Sigma | P3391 | |
Proteinase K | Fisher | BP1700-100 | |
RNase Out RNase inhibitor | Invitrogen | 10777-019 | 40 U/μl |
1 M NaCl | Fisher | BP358-212 | |
Sodium dodecyl sulfate | Fisher | BP166-500 | SDS |
1 M Tris-HCl | Fisher | BP153-500 | pH 7.4 |
Trizol | Invitrogen | 15596-026 | |
Vanadyl Ribonucleoside Complexes | New England Labs | S1402S | VRC |
Reagent Workup |
Prepare reagents in RNase/DNase-free, DEPC-treated glassware |
Polysome lysis Buffer |
100 mM KCl |
5 mM MgCl2 |
10 mM HEPES (pH 7.0) |
0.5% NP40 |
1 mM DTT |
100 units/ml RNase Out |
400 μM VRC |
Protease inhibitor cocktail tablet |
5 ml of Polysome lysis buffer |
Add 50 μl of 1 M HEPES (pH 7.0) |
500 μl of 1 M KCL |
25 μl of 1 M MgCl2 |
25 μl of NP40 |
4.7 ml RNase-DNase-free H2O |
50 μl of 1 M DTT |
12.5 μl of 100 U/ml RNase Out |
200 μl Protease inhibitor cocktail (dissolved according to manufacturer) |
10 μl 200 mM VRC (at time of use) |
NT2 Buffer |
50 mM Tris-HCl (pH 7.4) |
150 mM NaCl |
1 mM MgCl2 |
0.05% NP40 |
1 L of NT2 Buffer |
50 ml Tris (pH 7.4) |
30 ml 5 M NaCl |
1 ml 1 M MgCl2 |
500 μl NP40 |
820 ml RNase-DNase-free H2O |