A proportion of siRNAs cosediment with polyribosomes. Sucrose density gradient analysis of cytoplasmic extracts from Trypanosoma brucei cells expressing dsRNA for actin, MT40, and gfp. (A) Representative absorbance profile at 254 nm; the positions of the 80S monosome and polyribosomes are indicated. The positions of polyribosomes, 80S monosomes, and the 40S subunit were verified by examining the distribution of 18S and 28S ribosomal RNAs in the gradient fractions by denaturing agarose gel electrophoresis (data not shown). (B) Northern blot of small RNAs from sucrose density gradient fractions probed with a riboprobe representing the sense strand of actin dsRNA. The quantitation of siRNAs was carried out by phosphorimager analysis. The area corresponding to siRNAs in each lane was separately quantitated, and background was subtracted from each value. An average background value was determined by measuring several equivalent areas of the membrane above and below the siRNA bands. (C) Northern blot with a probe representing the sense strand of MT40 dsRNA. (D) Northern blot with a probe representing the sense strand of gfp dsRNA.
