Attomole sensitivity of Chemi-Northern detection. (A) EtBr-stained denaturing formaldehyde–MOPS agarose gel of 500 ng in vitro transcribed, purified synthetic Nluc mRNA and biotinylated Nluc antisense probe RNA (623 nt), confirmed the proper size, purity, and quality. RNA size marker is included with nucleotide (nt) lengths indicated on the left. We note that the migration of the Nluc probe RNA is slightly retarded due to the incorporation of biotinylated uridine. (B) Chemi-Northern detection of titrated synthetic Nluc mRNA at 6 sec exposure. The amount of the Nluc mRNA is indicated, with mass at the top and moles at the bottom. (C) Measured signal intensities in panel B are graphed relative to the amount of mRNA. Linear regression analysis was performed to assess the dynamic response, with the resulting line, equation, and coefficient of determination, R2. (D) Chemi-Northern detection of titrated synthetic Nluc mRNA at 6 sec exposures along with three samples containing 2.5 µg of total HCT116 RNA and synthetic Nluc RNA. The mass of synthetic Nluc mRNA in each lane is indicated at the top, and the number of moles is indicated at the bottom. (E) Measured signal intensities of the Nluc RNA (panel D) are graphed relative to the amount of Nluc RNA across the titration range (black circles). Linear regression analysis was performed to assess the dynamic response, with the resulting line, equation, and coefficient of determination, R2. Signal intensity for the three samples containing 0.35 ng of Nluc RNA in 2.5 µg of total HCT116 RNA is shown as light gray circles.
