Journal: Nature methods

Article Title: High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries

doi: 10.1038/nmeth.2078

Figure Lengend Snippet: ( a ) Schematic of the Sensor-seq assay. BdLV, bidirectional lentiviral vector. GFP, green fluorescent protein, NGFR, truncated nerve growth factor receptor. ( b ) Representative FACS plots from sorted THP1 cells. ( c ) Expression pattern of specific sensors as determined by Sensor-seq. Values are mean ± s.d.; n = 3. The P value was generated from a t-test comparison of GFP neg and NGFR + bins. ( d ) Representative FACS plots for individually transduced sensors; n = 3. ( e ) Comparative analysis of target suppression between monocyte, macrophage (MΦ), and kidney cell lines based on Sensor-seq. miRNA sensors were classified based on significant enrichment (≥2-fold, P < 0.05, t-test) in GFP neg :completely suppressed, GFP neg and GFP low : strongly suppressed, GFP low : suppressed, and GFP pos and GFP high : Not suppressed.

Article Snippet: Relative miRNA expression levels were confirmed by real-time PCR using the SABioscience miRNA qPCR whole genome array (Qiagen), and using Taqman miRNA assays for specific miRNAs.

Techniques: Plasmid Preparation, Expressing, Generated, Comparison

Journal: Nature methods

Article Title: High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries

doi: 10.1038/nmeth.2078

Figure Lengend Snippet: ( a ) miRNA expression levels in monocytes determined by deep sequencing. Values are the mean reads per million (RPM) ± s.d.; n = 3 shown for all miRNAs over 1 RPM. ( b ) Fold enrichment in the indicated bin over the total NGFR + population for each miRNA sensor, as determined by Sensor-seq. Values are mean ± s.d.; n = 3 ( c,d ) The concentration of each miRNA as a function of whether the its sensor was suppressed or not suppressed in ( c ) THP1 monocytes and ( d ) 293T embryonic kidney cells. A sensor was deemed suppressed if the frequency of the sensor was significantly enriched ( P <0.05 t-test) by 2-fold or more in GFP neg or GFP low bins compared to the total NGFR + population. Ind, individual miRNA. Fam, miRNA family.

Article Snippet: Relative miRNA expression levels were confirmed by real-time PCR using the SABioscience miRNA qPCR whole genome array (Qiagen), and using Taqman miRNA assays for specific miRNAs.

Techniques: Expressing, Sequencing, Concentration Assay

Journal: Nature methods

Article Title: High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries

doi: 10.1038/nmeth.2078

Figure Lengend Snippet: ( a ) Mean miRNA abundance as a function of the mean fold enrichment of corresponding PT or BT sensors in the GFP neg bin over the NGFR + population; n = 3. Note that a sensor not enriched in the GFP neg fraction may still be enriched in the GFP low fraction, and thus not all the points in this graph that are <2-fold correspond to a non-suppressive miRNA. ( b ) Ratio of the sum of predicted target transcript abundance to miRNA abundance for sensor library miRNAs in THP1 cells. n = 3. RPKM, average reads per kilobase of exon per million mapped reads. Only miRNAs expressed at >1,000 RPM are shown; miRNAs that were non-suppressive are highlighted. ( c ) Sensor-seq profiles of miR-16, miR-21 and miR-223 BT sensors in THP1 cells. Sensor frequencies are mean ± s.d.; n = 3. The frequency of each sensor in the total population of transduced cells is highlighted in red. The mean ± s.d. concentration of the corresponding miRNA is in parentheses; n = 3. ( d ) Representative FACS plots of THP1 monocytes 1 week after transduction with miR-16 or miR-21 BT sensors; n = 3. ( e ) Percentage of miRNAs in the nucleus relative to the entire cell for THP1 cells determined by quantitative PCR. Values are mean ± s.d.; n = 3.

Article Snippet: Relative miRNA expression levels were confirmed by real-time PCR using the SABioscience miRNA qPCR whole genome array (Qiagen), and using Taqman miRNA assays for specific miRNAs.

Techniques: Concentration Assay, Transduction, Real-time Polymerase Chain Reaction