Key Specifications Table
|Description||Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit|
|Overview||RNA-binding protein immunoprecipitation (RIP) is the RNA analog of the more well-known ChIP application (chromatin immunoprecipitation), which identifies DNA targets of DNA-binding proteins in an in-vivo cellular context. RIP can be used to identify specific RNA molecules (of many types) associated with specific nuclear or cytoplasmic binding proteins. These experiments involve immunoprecipitation of endogenously formed complexes of RNA-binding proteins and co-isolation of any RNA species associated with that RNA-binding protein. Purification of these RNA species allows interrogation and identification of mRNAs (and potentially non-coding RNAs associated with them) and can be directly measured using down stream applications including quantitative reverse transcription polymerase chain reaction (RT-PCR), microarray analysis (RIP-chip) and “deep-sequencing” or 2nd-generation sequencing based platforms (RIP-Seq).
Features & Benefits:
-Protein A/G magnetic beads, optimized to bind nucleic acid-protein immune complexes
-RNAse inhibitors and RNAse-free reagents
|Materials Required but Not Delivered||Magna Grip™ Rack 8 well ( 20-400) (Now Available!) or similar magnetic rack.|
|Background Information||Gene regulation plays a critical role in complex cellular processes such as development, differentiation, and cellular response to environmental changes. In addition to transcriptional regulation of gene expression by transcription factors, cells utilize post-transcriptional regulatory mechanisms. One such mechanism involves use of certain RNA-binding proteins (RBPs) to temporally and coordinately regulate the rate of mRNA translation of functionally related gene
products. While the regulation of gene expression by transcription factors has been well studied over time, the post-transcriptional regulation of mRNAs by RBPs and the role of non-coding RNAs in this process is a relatively nascent field that remains to be thoroughly explored.
|Presentation||Two boxes containing all necessary reagents to perform 12 individual RNA-binding protein immunoprecipitation (RIP) reactions.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Upon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 6 months from date of shipment when stored as directed.|
|Material Size||12 assays|
|Material Package||RIP Kit capacity: 12 RNA-binding protein immunoprecipitation assays|
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit SDS
|Reference overview||Application||Pub Med ID|
|LncRNA-PVT1 promotes pancreatic cancer cells proliferation and migration through acting as a molecular sponge to regulate miR-448.|
Zhao, L; Kong, H; Sun, H; Chen, Z; Chen, B; Zhou, M
J Cell Physiol 233 4044-4055 2018
The identification and characterization of long non-coding RNAs (lncRNAs) in diverse biological process has currently developed rapidly. LncRNA-PVT1, located adjacent to the MYC locus on chromosomal region 8q24, has been reported to be associated with many biological processes. However, the function and mechanism of PVT1 in pancreatic carcinoma (PC) is poorly understood. In this present study, we first measured the level of PVT1 in the PC cell lines and tissues by quantitative real-time PCR (qRT-PCR), and then employed loss-of-function and gain-of-function approaches to explore the association between PVT1 expression levels and PC cell proliferation/migration ability. Furthermore, bioinformatics analysis was utilized to show that PVT1 contains binding site for miR-448 and an inverse correlation between PVT1 and miR-448 was obtained in PC specimens. Additionally, dual luciferase reporter assay, RNA-binding protein immunoprecipitation (RIP) and applied biotin-avidin pulldown system were applied to further confirm that PVT1 directly bind with microRNA binding site harboring in the PVT1 sequence. Then, SERBP1 was identified as a target of miR-448 according to the gene expression array analysis of PC clinical samples. Together, we revealed that PVT1 functions as an endogenous "sponge" by competing for miR-448 binding to regulate the miRNA target SERBP1 and, therefore, promotes the proliferation and migration of PC cells.
|Linc00511 acts as a competing endogenous RNA to regulate VEGFA expression through sponging hsa-miR-29b-3p in pancreatic ductal adenocarcinoma.|
Zhao, X; Liu, Y; Li, Z; Zheng, S; Wang, Z; Li, W; Bi, Z; Li, L; Jiang, Y; Luo, Y; Lin, Q; Fu, Z; Rufu, C
J Cell Mol Med 22 655-667 2018
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Long non-coding RNAs (lncRNAs) are important regulators in pathological processes, yet their potential roles in PDAC are poorly understood. Here, we identify a fundamental role for a novel lincRNA, linc00511, in the progression of PDAC. Linc00511 levels in PDAC tissue specimens and cell lines were examined by quantitative real-time PCR. Corresponding adjacent non-neoplastic tissues were used as controls. The function of linc00511 in PDAC cell lines was determined by RNA interference approach in vitro and in vivo. Fluorescence in situ hybridization (FISH) was used to characterize linc00511 expression in PDAC cells. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were obtained from bioinformatic analysis, luciferase assays and RIP assays. The association between the linc00511/hsa-miR29b-3p axis and VEGFA was verified by Western blotting assay. Immunohistochemistry was performed to evaluate the expression of VEGFA in PDAC samples. The aberrant up-regulation of linc00511 was detected in PDAC cell lines and patient specimens compared with controls. An increase in linc00511 expression indicates the adverse clinical pathological characteristics and poor prognosis. Functionally, linc00511 depletion in PDAC cells decreased proliferation, migration, invasion and endothelial tube formation. Mechanistically, linc00511 could up-regulate VEGFA via its competing endogenous RNA (ceRNA) activity on hsa-miR-29b-3p. In summary, our results define an important axis controlling proliferation, invasion and tumour angiogenesis in PDAC. Linc00511 is a novel lncRNA that plays a significant regulatory role in the pathogenesis and progression of PDAC. Thus, Linc00511 represents a new prognostic biomarker to predict clinical outcome of PDAC patients after surgery and may serve as a potential therapeutic target for PDAC treatment.
|LncRNA KCNQ1OT1 ameliorates particle-induced osteolysis through inducing macrophage polarization by inhibiting miR-21a-5p.|
Gao, X; Ge, J; Li, W; Zhou, W; Xu, L
Biol Chem 399 375-386 2018
This study aimed to investigate the mechanism of lncRNA-KCNQ1OT1 on macrophage polarization to ameliorate particle-induced osteolysis. We used polymethylmethacrylate (PMMA) to induce primary bone marrow-derived macrophages (BMMs) obtained from mice and the RAW264.7 cell line, and found that the tumor necrosis factor-alpha (TNF-α) concentration and inducible nitric oxide synthase (iNOS) expression was increased, while interleukin (IL)-10 concentration and Arg1 expression were decreased in PMMA-induced cells. KCNQ1OT1 and IL-10 expression were both suppressed and miR-21a-5p expression was promoted in PMMA-induced cells. Overexpression of KCNQ1OT1 reversed the effect of PMMA on RAW264.7 cells, such as the reduced TNF-α concentration and iNOS expression, and increased IL-10 concentration and Arg1 expression in PMMA-induced cell transfected with pcDNA-KCNQ1OT1. The luciferase assay confirmed that IL-10 is a target of miR-21a-5p. RNA immunoprecipitation (RIP) and RNA pull-down experiments demonstrated that KCNQ1OT1 functions as a miR-21a-5p decoy. Thus, lncRNA KCNQ1OT1 induces M2 macrophage polarization to ameliorate particle-induced osteolysis by inhibiting miR-21a-5p.
|Neat1 regulates oxidized low-density lipoprotein-induced inflammation and lipid uptake in macrophages via paraspeckle formation.|
Huang-Fu, N; Cheng, JS; Wang, Y; Li, ZW; Wang, SH
Mol Med Rep 17 3092-3098 2018
Oxidized low-density lipoprotein (oxLDL) indu-ces macrophage inflammation and lipid uptake, and serves important roles in the development of atherosclerosis. The long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (neat1) has two isoforms; the longer isoform, neat1_2, mediates the formation of subnuclear structures called paraspeckles. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), western blotting and RNA protein immunoprecipitation (RIP), revealed that oxLDL induced paraspeckle formation in the THP‑1 cell line. Additionally, the nuclear factor‑κB and p38 pathways were observed to be involved in neat1 transcription. To investigate the role of paraspeckles in oxLDL‑induced macrophage inflammation and lipid uptake, macrophages were transfected with small interfering RNAs against NEAT1, NEAT1_2, non‑POU domain-containing octamer-binding (NONO) and splicing factor proline and glutamine rich prior to oxLDL incubation. In addition, inflammation‑associated pathways and scavenger receptors were analyzed by performing western blotting and RT‑qPCR. p65 phosphorylation and cluster of differentiation 36 (CD36) were demonstrated to serve roles in paraspeckle‑mediated inflammation and lipid uptake, respectively. To determine the underlying mechanism, RIP was preformed, which revealed that NONO binds CD36 mRNA to decrease its expression. In conclusion, oxLDL induced neat1_2‑mediated paraspeckle formation. Paraspeckles participate in oxLDL‑induced macrophage inflammation and lipid uptake by regulating p65 phosphorylation and CD36 mRNA.
|Auto- and cross-regulation of the hnRNPs D and DL.|
Kemmerer, K; Fischer, S; Weigand, JE
RNA 24 324-331 2018
HnRNP D, better known as AUF1, is an extensively studied protein that controls a variety of cellular pathways. Consequently, its expression has to be tightly regulated to prevent the onset of pathologies. In contrast, the cellular functions and regulation of its ubiquitously expressed paralog hnRNP DL are barely explored. Here, we present an intricate crosstalk between these two proteins. Both hnRNP D and DL are able to control their own expression by alternative splicing of cassette exons in their 3'UTRs. Exon inclusion produces mRNAs degraded by nonsense-mediated decay. Moreover, hnRNP D and DL control the expression of one another by the same mechanism. Thus, we identified two novel ways of how hnRNP D expression is controlled. The tight interconnection of expression control directly links hnRNP DL to hnRNP D-related diseases and emphasizes the importance of a systematic analysis of its cellular functions.
|TUG1 knockdown ameliorates atherosclerosis via up-regulating the expression of miR-133a target gene FGF1.|
Zhang, L; Cheng, H; Yue, Y; Li, S; Zhang, D; He, R
Cardiovasc Pathol 33 6-15 2018
Long non-coding RNAs (lncRNAs) have been revealed to participate in the pathological events associated with atherosclerosis. However, the exact role of lncRNA taurine-up-regulated gene 1 (TUG1) and its possible molecular mechanism in atherosclerosis remain unidentified.High-fat diet (HFD)-treated ApoE-/- mice were used as an in vivo model of atherosclerosis. Ox-LDL-induced macrophages and vascular smooth muscle cells (VSMCs) were employed as cell models of atherosclerosis. qRT-PCR was performed to detect the expression of TUG1 and miR-133a. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were analyzed by commercially available enzyme kits. Oil red O and hematoxylin and eosin (H&E) staining were conducted to examine atherosclerotic lesion. Luciferase reporter assay combined with RNA immunoprecipitation (RIP) was applied to confirm the interaction between TUG1, miR-133a and FGF1. Cell proliferation ability was determined by Cell Counting Kit-8 (CCK-8) assay and trypan blue dye exclusion test. Cell apoptosis was evaluated with TUNEL assay. Expression and production of inflammatory cytokines was measured with western blot and ELISA analysis.TUG1 expression was up-regulated in HFD-treated ApoE-/- mice, as well as in ox-LDL-induced RAW264.7 and MOVAS cells. TUG1 knockdown inhibited hyperlipidemia, decreased inflammatory response, and attenuated atherosclerotic lesion in HFD-treated ApoE-/- mice. TUG1 could function as a molecular sponge of miR-133a to suppress its expression. TUG1 overexpression accelerated cell growth, improved inflammatory factor expression, and inhibited apoptosis in ox-LDL-stimulated RAW264.7 and MOVAS cells, while this effect was abated after transfection with miR-133 mimic. Moreover, fibroblast growth factor 1 (FGF1) was identified as a direct target of miR-133a. Restored expression of FGF1 overturned the effect of miR-133a on cell proliferation, inflammatory factor secretion and apoptosis in ox-LDL-treated RAW264.7 and MOVAS cells. Finally, TUG1 was revealed to up-regulate FGF1 expression by sponging miR-133a.TUG1 knockdown ameliorates atherosclerosis by modulating FGF1 via miR-133a, raising the possibility of targeting TUG1 as an atheroprotective therapeutic strategy.
|AK098656, a Novel Vascular Smooth Muscle Cell-Dominant Long Noncoding RNA, Promotes Hypertension.|
Jin, L; Lin, X; Yang, L; Fan, X; Wang, W; Li, S; Li, J; Liu, X; Bao, M; Cui, X; Yang, J; Cui, Q; Geng, B; Cai, J
Hypertension 71 262-272 2018
Recent studies reported some long noncoding RNAs (lncRNAs)-mediated vascular smooth muscle cells (VSMCs) phenotypic switch, which was a common pathophysiological process of vascular diseases. However, whether human-specific expressed lncRNAs would modulate VSMCs phenotype and participate into the pathogenesis of essential hypertension remains unclear. By comparing the circulating lncRNAs expression profiles between hypertensive patients and healthy controls, we identified a lncRNA-AK098656, strongly upregulated in the plasma of hypertensive patients, and predominantly expressed in VSMCs. AK098656 promoted VSMCs synthetic phenotype evidenced by increasing VSMC proliferation and migration, elevating extracellular matrix proteins, whereas lowering contractile proteins. Furthermore, AK098656 was demonstrated to directly bind with the VSMCs-specific contractile protein, myosin heavy chain-11, and an essential component of extracellular matrix, fibronectin-1, and finally lowered these protein levels through protein degradation. AK098656 was also shown to bind with 26S proteasome non-ATPase regulatory subunit 11 and facilitated myosin heavy chain-11 to interact with this protein. In vivo, AK098656 transgenic rats showed spontaneous development of hypertension, with elevated VSMCs synthetic phenotype and narrowed resistant arteries. Transgenic rats also showed slight cardiac hypertrophy without other complications, which was similar with early pathophysiological changes of hypertension. All these data indicated AK098656 as a new human VSMC-dominant lncRNA, which could promote hypertension through accelerating contractile protein degradation, increasing VSMC synthetic phenotype, and finally narrowed resistance arteries.
|Long non-coding RNA DLEU1 predicts poor prognosis of gastric cancer and contributes to cell proliferation by epigenetically suppressing KLF2.|
Li, X; Li, Z; Liu, Z; Xiao, J; Yu, S; Song, Y
Cancer Gene Ther 25 58-67 2018
Currently, accumulating documents have paid great attention to the critical role of long non-coding RNAs. The long non-coding RNAs DLEU1 has been demonstrated to be dysregulated in many solid tumors and hematological malignancies. However, the detailed descriptions about its potential roles and molecular mechanism in gastric cancer (GC) are still blurry. As for our research, it was found out that DLEU1 was observably intensified in GC tissues and cell lines. And highly expressed DLEU1 was relevant to tumor size, advanced stage of pathology and lymph node metastasis in GC patients. Silenced DLEU1 obviously suppressed proliferation via leading to the cell cycle arrest and inducing cell apoptosis of GC. Furthermore, mechanistic experiments uncovered that DLEU1 could recruit LSD1 (lysine specific demethylase 1) to the promoter regions of KLF2 and then suppressed its transcription. In addition, rescue assays revealed that the oncogenic function mediated by DLEU1 in GC was partly by regulating KLF2. Collectively, our findings manifested that DLEU1 might serve as an oncogene in GC.
|LncRNA ZFAS1 promotes cell migration and invasion of fibroblast-like synoviocytes by suppression of miR-27a in rheumatoid arthritis.|
Ye, Y; Gao, X; Yang, N
Hum Cell 31 14-21 2018
Rheumatoid arthritis (RA) is a systemic and chronic inflammatory disease. Synoviocyte migration and invasion were found to be essential to the pathology of RA. Upregulation of long noncoding RNA ZFAS1 has been observed in cancers and promotes cell migration and invasion. To date, the functions and mechanisms of ZFAS1 in RA have not been revealed. In this study, we analyzed expression pattern of ZFAS1 in RA patients and found that ZFAS1 expression was increased in synovial tissue and fibroblast-like synoviocytes (FLS) from RA patients (RA-FLS) compared with that in healthy donors. Functional assays showed that silence of ZFAS1 suppressed RA-FLS migration and invasion, while overexpression of ZFAS1 showed the opposite effect. Further investigation demonstrated that ZFAS1 directly interacted with miR-27a and decreased miR-27a expression. ZFAS1 promotes RA-FLS migration and invasion in an miR-27a-dependent manner. Taken together, the present study provides the first evidence that ZFAS1 promotes cell migration and invasion through miR-27a in RA-FLS, suggesting that ZFAS1 may be an effective therapeutic target for RA patients.
|Effect of long non-coding RNA PVT1 on cell proliferation and migration in melanoma.|
Chen, L; Ma, D; Li, Y; Li, X; Zhao, L; Zhang, J; Song, Y
Int J Mol Med 41 1275-1282 2018
The present study aimed to investigate the potential role of the long non‑coding RNA (lncRNA) Pvt1 oncogene (non‑protein coding) (PVT1) in the progression and metastasis of malignant melanoma, and to reveal its possible molecular mechanisms. The expression of lncRNA PVT1 in melanoma tissues and adjacent normal skin from patients with melanoma, and in the melanoma A‑375 and sk‑mel‑5 cell lines, was analyzed using reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The effects of PVT1 expression on cell proliferation, the cell cycle, cell migration and cell invasion were analyzed using MTT assay, flow cytometry, Transwell and scratch assays, respectively. The interaction between PVT1 and enhancer of zeste homolog 2 (EZH2) in melanoma cells was analyzed using RNA immunoprecipitation (RIP) assay. The effect of PVT1 on microRNA‑200c (miR‑200c) expression was analyzed by chromatin immunoprecipitation (ChIP) assay. PVT1 was highly expressed in the melanoma tissues and cells. Silencing of PVT1 significantly inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G0/G1 stage. Additionally, PVT1 silencing significantly decreased the cyclin D1 expression in the melanoma cells. The expression of E‑cadherin was significantly increased and the expression of N‑cadherin and vimentin was significantly decreased in the PVT1‑silenced group. The RIP assay found that endogenous PVT1 was highly enriched by EZH2 RIP compared with that of the negative control. The ChIP assay revealed that the expression of miR‑200c was decreased significantly in the PVT1‑silenced group compared with the controls. Overall, the present study demonstrated that the lncRNA PVT1 may contribute to the tumorigenesis and metastasis of melanoma through binding to EZH2 and regulating the expression of miR‑200c. lncRNA PVT1 may serve as a potential target for the therapy of melanoma.
|RNA-Binding Protein Immunoprecipitation|
|White Paper - The Message in the Marks: Deciphering Cancer Epigenetics (EMD)|
|Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit|