RNA-biotin based pulldown assays for the detection of siRNA targeted genomic regions and siRNA directed histone modificationsChIP lysis buffer30ml final volume:1.5 ml 1M Hepes1.05ml 4M NaCl3ml 10% Triton X (Tx-100)300 l 10% NAD24.15ml H20 ChIP high salt buffer30ml final volume:1.5ml 1M Hepes3.75ml 4M NaCl3ml 10% Triton X (Tx-100)300 l 10% NAD21.45ml H20 ChIP wash buffer30ml final volume:150 l 2M Tris1.7ml 4.4M LiCl60 l 0.5M EDTA1.5ml 10% NAD1.5ml 10% NP-4025.09ml H20ChIP elution buffer20ml final volume:500 l 2M Tris2ml 10% SDS400 l 0.5M EDTA17.1ml H2Ocold lysis buffer1mM PMSF20units RNasin10mM Tris-HCl pH 7.410mM NaCl3mM MgCl20.1mM DTT0.5% NP40wash buffer10mM Tris pH 8.01mM EDTA0.5M NaClelution bufferTris-Cl pH 6.01mM EDTA2.0M NaCl0.5M MgCl2TBS-mod buffer50mM Tris HCl400mM NaCl pH 7.42x elute buffer10mM Tris-HCL pH 6.01mM EDTA2.0M NaClmodified lysis buffer0.5% NP-40300mM NaCl20mM Hepes pH 7.02mM MgCl2elution buffer II10mM Tris-HCl pH 6.01mM EDTA2.0M NaClBack to topAuthor NotesThe avidin/magnetic beads are first washed in lysis buffer (~500 l) followed by magnetic separation, aspiration, and then re-suspended in 100 l of lysis buffer. The 100 l bead slurry is then mixed with the siRNA/Flag-Tagged containing lysates and incubated at 4°C for an additional hour. Good washing is required to reduce the background. While MPG can deliver the siRNA directly to the nucleus it can also be cytotoxic at high volumes. For each wash incubate the samples on a rotating platform at room temperature for 3 minutes and then centrifuge at 14,000 RPM for 1 minute. Try to remove as much buffer as possible without aspirating the pelleted complex. Efficient washing is critical to reduced background. Include the input/starting sample saved for each sample from step 12 should also be reverse the cross-linked during this step. A glycogen carrier (Ambion) is recommended for visualization of the subsequent pellet. If assaying for promoter specific RNAs it is important to Dnase treat the elutes (Turbo DNA-free , Ambion) and then perform an RT reaction (+/- RT) followed by PCR with primers specific for the siRNA targeted promoter (Figure 2).Back to topReviewer CommentsReviewed by: Jeannie Lee, Department of Genetics (and Pathology), Harvard Medical School, Boston, MA, USA.The author describes a protocol to capture small RNA-associated proteins or DNA/RNA complexes. The procedures are based on immuno-affinity purification and chromatin immunoprecipitation (ChIP). The protocol could be an attractive means to verify small RNA associated complexes. Given its novelty and the timeliness of such a protocol, there would be much value in making the protocol available to the public. The protocol as described appears extremely difficult and may not be easy for the average investigator to reproduce, considering the sequential IP steps and the nature of small RNAs. Some of the concerns are noted below. The author uses the same buffer to extract nuclear fraction as that for cell lysis containing low salt (10mM NaCl). This would be a higher salt concentration buffer. Response: We use the same lysis buffer for both the initial lysis and then take the resultant pellet and re-lyse with the lysis buffer followed by sonication. This is not a very precise nucleus vs. cytoplasmic fractionation. Certainly upping the salt concentration could prove advantagous when assessing the 2 fractions. \"Flag-Input\" the author describes should be \"unbound\" The author should recover supernatant as \"Input\" before adding the anti-Flag-M2 beads. Response: This has been changed accordingly and the saved input is now in step 14 of Part-II. For preparation of genomic DNA by Qiagen DNeasy kit, guanidine hydrochloride would be used at the lysis step. Can DNA (or RNA)-siRNA hybrid maintain the formation under a denaturing condition without cross-link? Response: We have had success with and without the cross-link. Greater success is with number of cells. If one is trying to detect RNA/protein interactions using this method crosslinking should be used. If one is only trying to detect a promoter associated RNA then crosslinking is not necessary with numbers of cells, i.e. ~4×106. However, if one wants to detect the presence of a promoter associated RNA the easiest manner is via RT PCR with oligos designed to specifically amplify the promoter region. This of course is in retrospect as we first performed pulldown assays to detect these variant RNA species. The details will be published soon (Han et.al. PNAS, 2007, in press). We have changed this in the text accordingly. What is \"blocked ProteinA/Salmon Sperm\"? If this means beads like Protein A agarose or sepharose beads blocked with salmon sperm, the centrifuge to recover the beads would be too fast. It appears that the remainder of this section uses beads, but they are not mentioned. Response: We have removed the \"blocked\" statement as this seems misleading. The spin is correct we are trying to add the protein A/Salmon sperm to reduce the nonspecific background. We then spin a 14k to remove the bound fractions and retain the unbound as our elute for the antibody pulldown. Though clearly we could reduce the spin. In the bead recover in section (11) we have added the sepharose beads at step (11) and then wash accordingly. We have changed the text accordingly to make this section read more clearly. The author tries to isolate undigested genomic DNA binding with biotin-siRNA. How long is the average length of genomic DNA prepared by the Qiagen kit? Response: We have used this method with sonication and detected the promoter associated RNA but also without sonication. We do not know the average size of the resulting DNA following the Qiagen based extraction.Back to topFiguresFigure 1: A) Methodology for detection of flag-tagged DNMTs or HP1s associated with 5\' biotin labeled siRNA EF52. Schematic methodology is shown for detection from cell lysates of biotin labeled siRNAs in complex with flag-tagged DNMTs or HP1s (alpha, beta, or gamma). B) Triple immunoprecipitation assay for detection of H3K27me3+, flag-tagged DNMT3A, siRNA EF52 and the targeted EF1A promoter. Methodology is shown for performing first a ChIP for H3K27me3+ and then a Flag-immunoprecipitation followed by a biotin/avidin pulldown for biotin labeled siRNAs, or specifically the sense or antisense RNA. The resultant elutes are then utilized in PCR for the targeted EF1A promoter. Figure 2: 5\' biotin linked siRNA pull down assay to detect low-copy promoter specific RNAs. 293T or 293-R5-GFP cells (4×105/well, 6 well plate) or 4×106/10cm plate were transfected with 5\' biotin linked EF52 sense, antisense or sense/antisense RNAs (100nM). Twenty-four hours later the cultures were isolated, washed 1 time in 1x PBS, genomic DNA isolated (Qiagen Dneasy), and exposed to avidin magnetic beads (Dynal ). The final elutes, following the binding of the biotin linked RNAs with the avidin magnetic beads and washing, were then DNAse, RNAse H or RNAse A treated, RT (BioRad iScript) reacted and PCR amplified.Back to topReferencesKim, D.H., Villeneuve, L.M., Morris, K.V. and Rossi, J.J. (2006) Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells, Nature Structural and Molecular Biology, 13: 793-797. Matzke, M.A. and J.A. Birchler (2005) RNAi-mediated pathways in the nucleus, Nature Reviews Genetics, 6(1): 24-35. Morris, K.V. (2005) siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code, Cell and Molecular Life Sciences, 62(24): 3057-3066. Morris, K.V., Chan, S.W., Jacobsen, S.E. and Looney, D.J. (2004) Small interfering RNA-induced transcriptional gene silencing in human cells, Science, 305(5688): 1289-1292. Ting, A.H., Schuebel, K.E., Herman, J.G. and Baylin, S.B. (2005) Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation, Nature Genetics, 37(8): 906-910. Weinberg, M.S., Villeneuve, L.M., Ehsani, A., Amarzguioui, M., Aagaard, L., Chen, Z., Riggs, A.D., Rossi, J.J. and Morris, K.V. (2005) The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells, RNA, 12(2): 256-262.上一页[1][2]