Journal: Scientific Reports

Article Title: Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia

doi: 10.1038/s41598-017-00755-y

Figure Lengend Snippet: Detection of cells expressing BCR-ABL1 with PLA-flow. The assay employs a pair of oligonucleotide-conjugated antibodies (PLA probes) with affinity for the BCR and ABL1 domains of the fusion protein in fixed and permeabilized cells ( A ). The oligonucleotides on PLA probes remaining in close proximity after washes serve as templates for hybridization of two additional DNA oligonucleotides, guiding their ligation into DNA circles ( B ). After ligation, the DNA circles are locally amplified by rolling circle amplification (RCA) to generate RCA products that are visualized and detected by addition of complementary fluorophore-labeled oligonucleotides ( C ), followed by detection of labeled cells via flow cytometry.

Article Snippet: 0.5 U/µl phi-29 DNA polymerase (Fermentas) was mixed with RCA buffer (2x phi-29 DNA polymerase-buffer, pH 7.5 (Fermentas), 0.25 µg/µl BSA, 0.5 mM dNTP (Thermo Fisher Scientific)) and incubated for 90 min at 37 °C.

Techniques: Expressing, Proximity Ligation Assay, Flow Cytometry, Hybridization, Ligation, Amplification, Labeling, Cytometry

Journal: RNA

Article Title: Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3?-end

doi: 10.1261/rna.2068510

Figure Lengend Snippet: Target RNA conversion into a primer for RCA, the impact of E. coli RNase III. The reactions were carried out under the conditions described in Materials and Methods. The 5′-end-labeled ( A ) RNA2-PP1 (panel 1 ), ( B ) RNA2-PP2 (panel 1 ), and RNA1-PP1 (panel 2 ) hybrids were incubated with Phi29 DNA polymerase in the absence or presence of dNTP. ( A , panel 2 ) The experiments with E. coli RNase III were performed using RNA2-PP1 duplex. The RNA degradation and RCA products were analyzed by electrophoresis through denaturing 8% polyacrylamide gels. Different RNA-PP duplexes are shown above the gels. The radioactive label is indicated by the star. The contents of samples are shown above the gel lines. Reaction products are labeled as “ a ” for the 5′-end-labeled RNA1 hydrolysis product serving as an RNA primer for RCA and “ b ” for the 5′-end-labeled RCA product. ( C ) The reaction scheme represents the conversions of target RNA.

Article Snippet: BSA, DEPC-treated water, DNase I, DNA polymerases (Taq, Phi29), glycogen, RevertAid H Minus First Strand cDNA Synthesis Kit, REases (Mva1269I, LguI), Ribolock RNase inhibitor, T4 DNA ligase; PBS buffer (10×), SSC buffer (20×), Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Labeling, Incubation, Electrophoresis