I am working on a molecular immunology field and I perform an isolation of infected tissue from a model organism which further is proceeded with RNA isolation..... Now I want to know that can the RNA pull down assay be performed from the extracted RNA. If Yes, I would be oblighed if I can receive few papers regarding the protocol related to it.
Thank You
Dear Rajanya,
The following cover the answer to your question.
1-Methods Mol Biol. 2015;1206:87-95. doi: 10.1007/978-1-4939-1369-5_8.
RNA pulldown protocol for in vitro detection and identification of RNA-associated proteins.
Marín-Béjar O1, Huarte M.
Author information
Abstract
Recent advances in genomics have revealed that cells encode thousands of noncoding RNA molecules that do not require translation to proteins to perform their biological roles. Although very little is known about the mechanisms of function of noncoding RNAs, these undoubtedly involve the interaction with multiple cellular proteins. Here we describe a detailed RNA pulldown protocol for the in vitro detection of proteins specifically interacting with a long RNA molecule.
PMID:
25240889
[PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/pubmed/25240889
2-RNA pull-down assay
Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes
Miao-Chih Tsai, et al.
RNA pull-down assay using nuclear extract
1. Biotin-labeled RNAs were in vitro transcribed with the Biotin RNA Labeling Mix
(Roche) and T7 RNA polymerase (Promega), treated with RNase-free DNase I
(Promega) and purified with RNeasy Mini Kit (QIAGEN).
2. Biotin-HRP Northern blot was followed by manufacture’s manual (NorthernMax kit, Ambion) to demonstrate that all the RNAs are biotinylated and transcribed at the right size.
3. Three micrograms of biotinylated RNA was heated to 90°C for 2 minutes, put on ice for 2 minutes, supplied with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl, 10 mM MgCl2), and then shifted to room temperature (RT) for 20 minutes to allow proper secondary structure formation.
4. 107 HeLa cell pallets were resuspended in 2 ml PBS, 2 ml nuclear isolation buffer (1.28 M sucrose; 40 mM Tris-HCl pH 7.5; 20 mM MgCl2; 4% Triton X-100), and 6 ml water on ice for 20 min (with frequent mixing).
5. Nuclei were pelleted by centrifugation at 2,500 G for 15 min.
6. Nuclear pellet was resuspended in 1 ml RIP buffer (150 mM KCl, 25 mM Tris pH 7.4, 0.5 mM DTT, 0.5% NP40, 1 mM PMSF and protease Inhibitor (Roche Complete
Protease Inhibitor Cocktail Tablets)).
7. Resuspended nuclei were mechanically sheared using a dounce homogenizer with 15– 20 strokes.
8. Nuclear membrane and debris were pelleted by centrifugation at 13,000 RPM for 10 min.
9. Folded RNA was then mixed with 1mg of HeLa nuclear extract in RIP buffer and
incubated at RT for one hour.
10. Sixty microliters washed Streptavidin agarose beads (Invitrogen) were added to each binding reaction and further incubated at RT for one hour.
11. Beads were washed briefly five times in Handee spin columns (Pierce) and boiled in SDS buffer, and the retrieved protein was detected by standard western blot technique.
RNA pull-down assay using recombinant proteins
PRC2 complex were obtained from BPS Bioscience (5m: 51004; 3m: 51003). LSD1
complex (LSD1.com) was purified by tandem affinity purification from HeLa S3 cells as previously described (2). The complex contained known components (LSD1, BHC80, CoREST, HDACs, and BRAF35) in nearly stoichiometric amounts. Recombinant GSTLSD1 and 6xHis- CoREST were purified as described (2). Recombinant Flag-LSD1 was purified from Sf9 insect cells using M2-agarose affinity chromatography as previously described (5).
1. Biotin-labeled RNAs were in vitro transcribed with the Biotin RNA Labeling Mix
(Roche) and T7 RNA polymerase (Promega), treated with RNase-free DNase I
(Promega) and purified with RNeasy Mini Kit (QIAGEN).
2. Biotin-HRP Northern blot was followed by manufacture’s manual (NorthernMax kit, Ambion) to demonstrate that all the RNAs are biotinylated and transcribed at the right size.
3. Three micrograms of biotinylated RNA was heated to 90°C for 2 minutes, put on ice for 2 minutes, supplied with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl, 10 mM MgCl2), and then shifted to room temperature (RT) for 20 minutes to allow proper secondary structure formation.
4. 107 HeLa cell pallets were resuspended in 2 ml PBS, 2 ml nuclear isolation buffer (1.28 M sucrose; 40 mM Tris-HCl pH 7.5; 20 mM MgCl2; 4% Triton X-100), and 6 ml water on ice for 20 min (with frequent mixing).
5. Nuclei were pelleted by centrifugation at 2,500 G for 15 min.
6. Nuclear pellet was resuspended in 1 ml RIP buffer (150 mM KCl, 25 mM Tris pH 7.4, 0.5 mM DTT, 0.5% NP40, 1 mM PMSF and protease Inhibitor (Roche Complete
Protease Inhibitor Cocktail Tablets)).
7. Resuspended nuclei were mechanically sheared using a dounce homogenizer with 15– 20 strokes.
8. Nuclear membrane and debris were pelleted by centrifugation at 13,000 RPM for 10 min.
9. 0.1 microgram of biotinylated RNA was incubated with different amounts of indicated proteins (2.5 and 5 µg of PRC2-5m and PRC2-3m; 3 and 5 µl of LSD1.com; 0.4 and 4 µg of GST-LSD1; 1 and 5 µg of Flag-LSD1; 0.4 and 4 µg of His-CoREST). 13. RNA and proteins were added in 200 µl binding buffer (50 mM TrisCl 7.9, 10% Glycerol, 100 mM KCl, 5 mM MgCl2, 10 mM β-ME 0.1% NP- 40) and incubated at RT for one hour.
10. For Fig S3B experiment, 3 µl DNase I (Invitrogen, 18047- 019) was added incubated at 37 °C for 30 minutes, and then follow the IP process.
11. Twenty microliters washed Streptavidin agarose beads (Invitrogen) were added to each binding reaction and further incubated at RT for 30 minutes.
12. Beads were washed briefly five times in Handee spin columns (Pierce) and boiled in SDS buffer, and the retrieved protein was detected by standard Western blot technique.
http://changlab.stanford.edu/RNA_pull-down_assay.pdf
3-Profiling direct mRNA-microRNA interactions using synthetic
biotinylated microRNA-duplexes
Shivangi Wani and Nicole Cloonan
QIMR Berghofer Medical Research Institute, Genomic Biology Lab, 300 Herston Rd, Herston, QLD, Australia, 4068. Correspondence should be
addressed to N.C ([email protected]).
MicroRNAs (miRNAs) are predominantly negative regulators of gene expression that act through the RNA-induced Silencing Complex (RISC) to suppress the translation of protein coding mRNAs. Despite intense study of these
regulatory molecules, the specific molecular functions of most miRNAs remain unknown, largely due to the challenge of accurately identifying miRNA targets. Reporter gene assays can determine direct interactions, but are laborious
and do not scale to genome-wide screens. Genomic scale methods such as HITS-CLIP do not preserve the direct interactions, and rely on computationally derived predictions of interactions that are plagued by high false positive
rates. Here we describe a protocol for the isolation of direct targets of a mature miRNA, using synthetic biotinylated miRNA duplexes. This approach allows sensitive and specific detection of miRNA-mRNA interactions, isolating high
quality mRNA suitable for analysis by microarray or RNAseq.
http://biorxiv.org/content/biorxiv/early/2014/05/22/005439.full.pdfl,
Hoping this will be helpful,
Rafik
Thank you very much Rafik for all the information. However in the first paper you mention I notice that
"The sequence of the RNA of interest should be cloned into a plasmid under control of the T7 promoter, to allow in vitro transcription with T7 polymerase.
For in vitro transcription, the plasmid should be linearized by digestion with a restriction enzyme cutting right after the 3′ end of the RNA sequence .
I though instead that I would have to cut before the T7 promoter. Is it right?. The insert I want to transcribe is insert in Not BamH1 of pcDNA3.1 . I though that I have to linearize the plasmid by NdeI just before the T7 promoter located before my insert . Am I right??
Thank you in advance.
Evelyne
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