miR-506 mimics or miRNA was transfected into HCCC-9810 and CCLP1 cells. And the cells were lysed and collected in a RIP lysis buffer kit (Millipore, USA). Human anti-Ago2 (Millipore, USA) or mouse anti-IgG (Millipore, USA) were then conjugated with RNAs magnetic beads. Then the expression levels of purified RNA were determined by qRT-PCR.
Rip lysis buffer kit
Manufactured by Merck Group
Sourced in United States
The RIP lysis buffer kit is a laboratory reagent used for the extraction and isolation of ribonucleoprotein (RNP) complexes from cells or tissues. The kit provides a buffer solution designed to gently lyse cells and preserve the integrity of RNP complexes, allowing for their subsequent analysis or purification.
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7 protocols using rip lysis buffer kit
1
Investigating miR-506 Regulation by RIP
2
RNA Immunoprecipitation Assay Protocol
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RIP lysis buffer kit (Millipore) was used for RIP assay. Briefly, LCA cells were lysed in RIP lysate buffer, and RNA was precipitated with Anti-AGO2 (Millipore) and anti-IgG (Millipore). TRIzol reagent was used to purify the immunoprecipitated RNA, and the gene expression was detected using RT-qPCR.
3
RNA Immunoprecipitation Protocol
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RIP lysis buffer kit (Millipore, Billerica, MA, USA) was used for RIP experiments. Summing up, HeLa cells were lysed in RIP lysis buffer, and RNA was precipitated with anti-AGO2 (Millipore, Billerica, MA, USA) and anti-IgG (Millipore, Billerica, MA, USA). TRIzol reagent was used to purify the immunoprecipitated RNA, and RT-qPCR was used to detect the gene expression.
4
Ago2 RNA Immunoprecipitation and Analysis
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Cells were lysed in a RNA immunoprecipitation (RIP) lysis buffer kit (Millipore), and then the cell extract was incubated with anti-Ago2 (Millipore) or anti-immunoglobulin G (Millipore) conjugated with magnetic beads at 4oC for 6 hours. The antibody-beads complex was resuspended in proteinase K Buffer. After removing the precipitation, RNA in the supernatant was purified. At last, quantitative reverse transcription real-time polymerase chain reaction (RT-qPCR) was applied to detect the enrichment of purified RNAs. The experiment was conducted at least three times.
5
RIP Assay for AGO2-Bound RNA
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A RIP lysis buffer kit (Millipore Corp, Billerica, MA, USA) was utilized for RIP experiments. In brief, MC3T3-E1 cells were lysed in RIP lysis buffer and incubated with anti-AGO2 (Millipore Corp) and anti-IgG (Millipore Corp)-coupled A/G agarose particles. The precipitated RNA was isolated using TRIzol reagents, and gene expression was determined using RT-qPCR.
6
RIP Assay for AGO2 Interactome
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A RIP lysis buffer kit (Millipore Corp, Billerica, MA, USA) was utilized for RIP experiments. Brief, MC3T3-E1 cells were lysed in RIP lysis buffer and incubated with anti-AGO2 (Millipore Corp) and anti-IgG (Millipore Corp)-coupled A/G agarose particles. The precipitated RNA was isolated using TRIzoL reagents, and gene expression was determined using RT-qPCR.
7
Luciferase Assay and RNA Immunoprecipitation for miR-506 Interactions
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The sequences containing the wild-type (WT) or mutated (MUT) region DDX5 and linc00473 were synthesized by GenePharma (Shanghai, China) and inserted into a pmirGLO-Report luciferase vector. For the luciferase reporter assay, miR-506 mimics and the respective reporter plasmids were transfected into cells using Lipofectamine 3000 according to the manufacturer's protocol. After 24 hours, the Renilla and Fire y luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
RNA-binding protein immunoprecipitation (RIP) assay miR-506 mimics or miRNA was transfected into HCCC-9810 and CCLP1 cells. And the cells were lysed and collected in a RIP lysis buffer kit (Millipore, USA). Human anti-Ago2 (Millipore, USA) or mouse anti-IgG (Millipore, USA) were then conjugated with RNAs magnetic beads. Then the expression levels of puri ed RNA were determined by qRT-PCR.
The immunohistochemistry (IHC) assay IHC analysis was performed as previously described [16] . For this staining, after heat-induced epitope retrieval, para n-embedded sections were incubated with 3% H
RNA-binding protein immunoprecipitation (RIP) assay miR-506 mimics or miRNA was transfected into HCCC-9810 and CCLP1 cells. And the cells were lysed and collected in a RIP lysis buffer kit (Millipore, USA). Human anti-Ago2 (Millipore, USA) or mouse anti-IgG (Millipore, USA) were then conjugated with RNAs magnetic beads. Then the expression levels of puri ed RNA were determined by qRT-PCR.
The immunohistochemistry (IHC) assay IHC analysis was performed as previously described [16] . For this staining, after heat-induced epitope retrieval, para n-embedded sections were incubated with 3% H
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