Dynamic response range of mRNA detection by Chemi-Northern using biotinylated RNA probes. HCT116 cells were transfected with increasing amounts (250, 500, 1000, and 2000 ng) of Nluc reporter plasmid, as indicated at the top. The “0” ng condition indicates “mock” transfected cells. A total of 5 µg of purified cellular RNA was loaded into each lane of the denaturing formaldehyde–MOPS agarose gel and detected via Chemi-Northern using 50 ng/mL (A) or 500 ng/mL (B) of biotinylated antisense Nluc RNA probe with 8% biotinylated nucleotides. Chemi-Northern blot of expressed Nluc mRNA (639 nt + pA tail) at 1 sec exposure time is shown in the upper panels, and EtBr stain of rRNA (18S, 1869 nt and 28S rRNA, 5070 nt), serving as a loading control and indication of RNA integrity, are shown in the lower panels. (C) Graph showing the linear relationship between the transfected reporter (x-axis) and signal intensity (y-axis) of Chemi-Northern blot at two different probe concentrations (50 and 500 ng/mL). The linear regression equation, y = mx + b, and coefficient of determination, R2 , are reported. All blots were quantified using AzureSpot Pro and graphs were created using GraphPad Prism software. (D) HCT116 cells were transfected with 1.25 µg of Nluc reporter plasmid, and the purified total RNA was titrated (50, 500, 1000, 2500, and 5000 ng) and expressed. Nluc mRNA was detected at 1 sec exposure by Chemi-Northern blot (upper panel) and EtBr stain (lower panel). (E) Quantitation and linear fit between total RNA mass (ng, x-axis) and signal intensity (y-axis). RNA was analyzed from Drosophila DL1 cells (F, G) or D.mel-2 cells (H, I) transfected with Nluc (806 nt + pA tail) or Fluc (1773 nt + pA tail) reporters using Chemi-Northern. The Nluc and Fluc probes each had 8% biotinylated nucleotides in this experiment. Purified total cellular RNA was titrated as indicated at the top of the gels. Note that in panel F, the bottom panel shows the Drosophila rRNA species stained with EtBr, of which the 28S rRNA (3945 nt) is naturally processed into two fragments (1787 and 2112 nt), whereas the 18S rRNA is 1995 nt, as previously documented (Long and Dawid 1980; Tautz et al. 1988). (G, I) Quantitation of signal intensity relative to the amount of total cellular RNA analyzed.
