Journal: Stem cells (Dayton, Ohio)
Article Title: Genome-Wide Analysis of miRNA-mRNA Interactions in Marrow Stromal Cells
Figure Lengend Snippet: Ago HITS-CLIP analysis of stromal and endothelial cells. (A): Basic experimental scheme of Ago HITS-CLIP as adapted from Chi et al. UV cross-linked cells are lyzed and treated with RNase to reduce the length of mRNA fragments. Ago is immune-precipitated with monoclonal anti-Ago antibody 2A8, P32-labeled 3′ RNA linker ligated (on-bead), and resolved by SDS-PAGE electrophoresis. The protein-RNA-complex is transferred to nitrocellulose and visualized by autoradiography. In the representative autoradiograph shown, a major band is seen ~110 kD and a more diffuse band at ~130 kD. The RNA-protein complex in the 110–130 kD region is isolated and a 5′ RNA linker is ligated. The isolated RNA is reverse transcribed and PCR amplified with Illumina-compatible primers to generate a cDNA library (typical agarose gel shown). The cDNA libraries are then gel purified and sequenced on Illumina GA IIx and resultant bioinformatically analyzed. (B): Distribution of aligned reads from HS27A samples to different gene annotations. (C): Correlation of consensus peaks between HS5 and HS27A. Coefficient of determination ( R 2 determined by corrplot [R-package]). (D): Correlation of consensus peaks between HS5 and hMSC. (E): Correlation of consensus peaks between HS27A and hMSC. (F): Correlation of consensus peaks between TrHBMEC and HUVEC. (G): Overlap of genes enriched by consensus peaks between stromal cells (HS5, HS27A, and hMSC). (H): Overlap of genes enriched by consensus peaks between endothelial cells (TrHBMEC and HUVEC). Abbreviations: hMSC, human mesenchymal stromal cell; HUVEC, human umbilical vein endothelial cell; TrHBMEC, transformed human bone marrow endothelial cells; UV, ultraviolet.
Article Snippet: Sequencing was performed on an Illumina Genome Analyzer IIx (single end 50 base pair reads).
Techniques: Cross-linking Immunoprecipitation, Labeling, SDS Page, Electrophoresis, Autoradiography, Isolation, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Agarose Gel Electrophoresis, Purification, Transformation Assay