Short Report
A useful approach to total analysis of RISC-associated RNA
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* Corresponding author: Yukinobu Hayashida hayashida.yukinobu@wako-chem.co.jp
- † Equal contributors
1 Genome Research Laboratories, Wako Pure Chemical Industries, Ltd. Takada 6-1, Amagasaki, Hyogo 661-0963, Japan
2 Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan
BMC Research Notes 2009, 2:169 doi:10.1186/1756-0500-2-169
Published: 26 August 2009Additional files
Additional file 1:
Anti-human Ago2 monoclonal antibody (4G8) specifically immunoprecipitates human Ago2 and associated small RNA. A: SDS-PAGE pattern of immunoprecipitates. Lysates of human cell lines (HeLa, HepG2, HEK293, and THP-1) and a mouse cell line (P388D1) were immunoprecipitated with anti-hAgo2 (4G8). Proteins were analyzed by SDS-PAGE and subjected to silver staining or western blotting with anti-hAgo2 (4G8). Half of the immunoprecipitated protein prepared from 1 × 107 cells was loaded onto an SDS-polyacrylamide gel. IgG h.c. and l.c. indicate heavy chain and light chain of the antibody molecule, respectively. B: Recovery of small RNA from immunoprecipitates. Immunoprecipitated small RNAs were analyzed by Urea-PAGE and silver stained. The RNA fraction prepared from half of the immunoprecipitate from 1 × 107 cells was loaded onto Urea-polyacrylamide gel.
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Additional file 2:
Comparison of miRNA populations of total RNA and immunoprecipitated RNA in HeLa. Microarray analysis of miRNA was carried out at LC science inc. Immunopurified RNA and total RNA from HeLa cells were labeled with Cy3 and Cy5. Samples were hybridized to μParaflo® microfluidics chip with each of the detection probes containing a nucleotide sequence of coding segment complementary to human 474 microRNA sequences(miRBase ver.9.0).
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Additional file 3:
Schematic representation of the cloning protocol for immunoprecipitated mRNA. The protocol is described in detail in Materials and methods (Additional file 1).
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Additional file 4:
The list of the cDNA clones derived from mRNA deduced by a BLAST search in HeLa.
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Additional file 5:
Homology search for the predicted free Alu RNA clones in HeLa by GENETYX®.
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Additional file 6:
The list of the cDNA clones derived from mRNA deduced by a BLAST search in miR-122 and GL3 transfected HepG2.
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