Detection of low to moderately expressed endogenous mRNAs with increased sensitivity. (A) Total RNA was purified from HCT116 cells and titrated (50, 100, 1000, 2500, and 5000 ng) onto a formaldehyde agarose gel followed by Chemi-Northern blotting using biotinylated RNA antisense probes (19% biotinylated nucleotides) synthesized using a 3:1 bioU:U ratio to detect moderately expressed endogenous B2M mRNA (943nt + pA tail). The exposure time was 7 sec. EtBr staining of rRNAs is shown in the lower panel. (B) Graph of quantitation of panel A showing linear fit of the total RNA mass (ng) versus signal intensity of the B2M. (C) The lowly expressed GRIP and coiled-coil domain containing 2 (GCC2) mRNA (6909nt + pA tail) was detected with a 10 sec exposure with a probe containing 25% biotinylated nucleotides. (D) Graph of quantitation of GCC2 mRNA in panel C. (E) The lowly expressed DIS3-like exosome 3′–5′ exoribonuclease (DIS3L) mRNA (3780 nt + pA tail) was detected at 10 sec exposure using a probe with 23% biotinylated nucleotides. (F) Graph of quantitation of DIS3L mRNA in panel E.
