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
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