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Figure 1 | BMC Biology

Figure 1

From: Control of chicken CR1 retrotransposons is independent of Dicer-mediated RNA interference pathway

Figure 1

Conditional loss of Dicer function in chicken DT40 cell line. (A) Western blot analysis of Dicer-deficient whole-cell extracts with anti-Dicer antibody at the indicated times after addition of 2 μg/ml doxycycline (Dox). Equal amounts of extracts (approximately 20 μg) were resolved by SDS-PAGE and equal loading was confirmed by western blot analysis with an anti-β-actin antibody. (B) Quantitative real-time RT-PCR analysis of Dicer mRNA was determined in Dicer+ cells (Dox-) and Dicer-deficient cells (Dox+). Plotted are values (in arbitrary units) for abundance of Dicer transcripts normalized to levels of chicken β-actin transcripts, a housekeeping gene. (C) The L1 transcripts derived from L1 elements of human chromosome 21 were detected in Dicer-deficient cells by strand-specific RT-PCR with primers specific for the L1's ORF1 and ORF2 sequences. Chicken β-actin served as an internal control. (D) Quantitative real-time RT-PCR analysis of human L1 mRNA was determined in the presence (Dicer-deficient) or absence of Dox (control cells). Relative fold change of L1's ORF1 and ORF2 mRNA levels after depletion of Dicer for 48 h was determined by normalizing the data with chicken β-actin gene. Error bars show s.d (n = 4). (E) Northern blot analysis of the human L1-specific siRNAs in chicken DT40 cells. The small RNAs extracted from control DT40 (Dox-) or Dicer-depleted DT40 cells (Dox+) were probed with L1 sequence and its signal density was quantified (see in insert). Signal density of control value was considered as 100%.

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