Figure 3.
Analyses of satellite repeat transcripts in ES cells in the presence or absence of Dicer. (A) Quantitative real-time RT–PCR was performed to determine the relative abundance of transcripts from pericentric major satellite repeats in control (Dcrneo/+, gray bars) and mutant (DcrΔ/Δ, black bars) ES cells. Total RNA samples were treated with DNase I prior to reverse transcription. Plotted are values (in arbitrary units) for abundance of major satellite transcripts normalized to levels of hprt transcripts, a housekeeping gene. ES cells were either untreated or treated with increasing concentrations of the histone deactylase inhibitor, TSA. (B) Semiquantitative RT–PCR was performed to determine the relative abundance of transcripts from centromeric minor satellite repeats in control (neo/+ and neo/neo) and mutant (Δ/Δ) ES cells. Digital photographs (negative image) of agarose gel analyses of the RT–PCR products for minor satellite and β-actin transcripts are shown. Total RNA samples were treated with DNase I prior to reverse transcription. Equivalent amounts of reverse-transcribed RNA were used for each PCR reaction as determined by the abundance of hprt transcripts, which was obtained by quantitative real-time RT–PCR (data not shown). (C) Total RNA from wild-type ES cells was either untreated (–) or treated (+) with RNase ONE and resolved in a denaturing 15% acrylamide gel. Shown in the left panel is the gel stained with ethidium bromide (EtBr). In the right panel, the gel was blotted and probed with a radiolabeled probe specific for minor satellite repeats (pMR150). A radiolabeled Decade marker (M) consisting of 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, and 150-nt RNA species is included. (D) Total RNA was size fractionated using mirVana glass fiber filters and equivalent amounts of the small RNA fraction (<200 nt) were resolved in a denaturing 15% acrylamide gel. Shown in the left panel is the gel stained with EtBr loaded with short RNAs from wild-type (neo/+) or mutant (Δ/Δ) ES cells. In the right panel, the gel was used for a Northern blot hybridized with a probe specific for minor satellite repeats (pMR150). A radiolabeled RNA Decade ladder (M) is also included. (E) Total RNA was size fractionated using mirVana glass fiber filters, and equivalent amounts of the long RNA fraction (>200 nt) were resolved in a denaturing 10% acrylamide gel and analyzed by Northern blot as in C and D. The EtBr-stained gel is shown below.