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. 2009 Jan 30;5(1):e1000354. doi: 10.1371/journal.pgen.1000354

Figure 3. FL-L1b transcription and RNA-ChIP analysis.

Figure 3

(A) A schematic diagram showing the FL-L1b target sites for the three independent primer sets (FL-L1b.1, FL-L1b.2, and FL-L1b.3; together spanning a genomic region of 415 bp) used in RT-PCR assays. (B) Positive transcription of FL-L1b was seen with all 3 primer sets in CHOK1-N10 and CHOK1-M10 cell lines, but not in CHOK1 #8 cell line after 40 cycles of RT-PCR amplification. CHOK1 #8, CHOK1-N10 and CHOK1-M10 were hamster-human monochromosomal hybrid lines containing unrelated normal human chromosome 10, progenitor normal human chromosome 10, and mardel(10) chromosome, respectively. The transcription of positive control β-actin gene was detected in all the cell lines. ‘¢’ = genomic DNA control. (C) Quantitative RT-PCR results (mean for n = 3, with standard error of the mean, SEM), indicating no significant difference in FL-L1b transcription in CHOK10-N10 and CHOK1-M10. Relative FL-L1b transcription levels in CHOK1-N10 and CHOK1-M10 were compared to that of CHOK1 #8, which was used as a normalization control. ΔCT = CT [test segment]−CT [β-actin control]. (D) RNA-ChIP-qPCR analysis. ChIP was performed using an anti-CENP-A antibody followed by quantitative RT-PCR analysis. FL-L1b RNA was significantly enriched (P<0.05) in the CENP-A-bound fraction in CHOK1-M10 but not in CHOK1-N10. None of the negative controls (18S rDNA, 5S rDNA, and β-actin ACTB RNAs) was enriched in the precipitated fractions. Relative binding values (mean for n = 5, with SEM) on the Y-axis represent the fold-enrichment of FL-L1b RNA CHOK1-M10 compared to that of CHOK1-N10.