Analysis of H2B, H3, and H4 mRNAs by Northern blot and gene-specific quantification of histone mRNA levels using the NanoString technology. (A,B) HeLa cells were tranfected with control, I6.1, I6.3, or MIF4GD siRNAs, and total RNAs were purified and separated on a polyacrylamide gel. The gel was stained with ethidium bromide, and the signal corresponding to 5S rRNA is shown as the loading control (bottom panels). After transfer to nylon membranes, hybridization was performed with an oligonucleotide corresponding to a region highly conserved among H3 genes on the antisense strand (A, middle panel). The membrane was stripped and hybridized again with an H2B probe (A, top panel). The same experiment with another gel was performed using an H4 probe (B, top panel). Numbers below the blot are the fold change with respect to control siRNAs after quantification and normalization with respect to 5S RNA. (C) HeLa cells were transfected with control, I6.1, or I6.3 siRNAs. Total RNAs were prepared and quantified with the nCounter apparatus using probes specific to the histone genes indicated, as well as of EIF3E and MIF4GD mRNA. The graph represents the mean of either two (I6.1, dark gray) or three (I6.3, light gray) independent experiments as ratio with respect to control siRNA; error bars indicate the standard deviation.
