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Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification.

van Bakel H, van Werven FJ, Radonjic M, Brok MO, van Leenen D, Holstege FC, Timmers HT - Nucleic Acids Res. (2008)

Bottom Line: Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis.In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p.The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.

ABSTRACT
Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) is a powerful technique to detect in vivo protein-DNA interactions. Due to low yields, ChIP assays of transcription factors generally require amplification of immunoprecipitated genomic DNA. Here, we present an adapted linear amplification method that involves two rounds of T7 RNA polymerase amplification (double-T7). Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis. In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p. The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR. Both LM-PCR and double-T7 identified strongly bound genomic regions, but the double-T7 method increased sensitivity and specificity to allow detection of weaker binding sites.

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Comparison of significantly Gsm1p-bound regions after double-round T7 or LM-PCR amplification. The Gsm1p-bound regions identified after double-round T7 amplification (double-T7) and LM-PCR were compared after ANOVA analysis with high- (q < 0.05 and binding ratio>2) (A–D) or low-stringency (q < 0.05 and binding ratio > 1.5) cutoffs (E–H). Venn diagrams (A and E) indicate the degree of overlap between the binding peaks identified after double-T7 or LM-PCR amplification. Average binding profiles were determined for gene regions with significant binding on the ORF or 800 bp of promoter region as indicated (B–D, F–H). Averaged background binding profiles derived from randomly selected sets of genes of similar size are shown as dashed lines. (I) Direct comparison of the maximal peak height after double-T7 (red) or LM-PCR (blue) for all overlapping binding peaks identified in (E).
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Figure 4: Comparison of significantly Gsm1p-bound regions after double-round T7 or LM-PCR amplification. The Gsm1p-bound regions identified after double-round T7 amplification (double-T7) and LM-PCR were compared after ANOVA analysis with high- (q < 0.05 and binding ratio>2) (A–D) or low-stringency (q < 0.05 and binding ratio > 1.5) cutoffs (E–H). Venn diagrams (A and E) indicate the degree of overlap between the binding peaks identified after double-T7 or LM-PCR amplification. Average binding profiles were determined for gene regions with significant binding on the ORF or 800 bp of promoter region as indicated (B–D, F–H). Averaged background binding profiles derived from randomly selected sets of genes of similar size are shown as dashed lines. (I) Direct comparison of the maximal peak height after double-T7 (red) or LM-PCR (blue) for all overlapping binding peaks identified in (E).

Mentions: The reduction in noise by the double-T7 method is expected to result in an improved detection of Gsm1p-binding sites. To test this we first analyzed the ChIP-chip data using both stringent and less stringent statistical tests. The stringent analysis (ANOVA q < 0.05 and binding ratio ≥ 2) identified 140 genomic-binding events for Gsm1p using double-T7, compared to 66 events for LM-PCR (Figure 4A). There is considerable agreement between the two methods, with 68% of LM-PCR-identified binding events overlapping double-T7 identified regions.Figure 4.


Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification.

van Bakel H, van Werven FJ, Radonjic M, Brok MO, van Leenen D, Holstege FC, Timmers HT - Nucleic Acids Res. (2008)

Comparison of significantly Gsm1p-bound regions after double-round T7 or LM-PCR amplification. The Gsm1p-bound regions identified after double-round T7 amplification (double-T7) and LM-PCR were compared after ANOVA analysis with high- (q < 0.05 and binding ratio>2) (A–D) or low-stringency (q < 0.05 and binding ratio > 1.5) cutoffs (E–H). Venn diagrams (A and E) indicate the degree of overlap between the binding peaks identified after double-T7 or LM-PCR amplification. Average binding profiles were determined for gene regions with significant binding on the ORF or 800 bp of promoter region as indicated (B–D, F–H). Averaged background binding profiles derived from randomly selected sets of genes of similar size are shown as dashed lines. (I) Direct comparison of the maximal peak height after double-T7 (red) or LM-PCR (blue) for all overlapping binding peaks identified in (E).
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Figure 4: Comparison of significantly Gsm1p-bound regions after double-round T7 or LM-PCR amplification. The Gsm1p-bound regions identified after double-round T7 amplification (double-T7) and LM-PCR were compared after ANOVA analysis with high- (q < 0.05 and binding ratio>2) (A–D) or low-stringency (q < 0.05 and binding ratio > 1.5) cutoffs (E–H). Venn diagrams (A and E) indicate the degree of overlap between the binding peaks identified after double-T7 or LM-PCR amplification. Average binding profiles were determined for gene regions with significant binding on the ORF or 800 bp of promoter region as indicated (B–D, F–H). Averaged background binding profiles derived from randomly selected sets of genes of similar size are shown as dashed lines. (I) Direct comparison of the maximal peak height after double-T7 (red) or LM-PCR (blue) for all overlapping binding peaks identified in (E).
Mentions: The reduction in noise by the double-T7 method is expected to result in an improved detection of Gsm1p-binding sites. To test this we first analyzed the ChIP-chip data using both stringent and less stringent statistical tests. The stringent analysis (ANOVA q < 0.05 and binding ratio ≥ 2) identified 140 genomic-binding events for Gsm1p using double-T7, compared to 66 events for LM-PCR (Figure 4A). There is considerable agreement between the two methods, with 68% of LM-PCR-identified binding events overlapping double-T7 identified regions.Figure 4.

Bottom Line: Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis.In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p.The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.

ABSTRACT
Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) is a powerful technique to detect in vivo protein-DNA interactions. Due to low yields, ChIP assays of transcription factors generally require amplification of immunoprecipitated genomic DNA. Here, we present an adapted linear amplification method that involves two rounds of T7 RNA polymerase amplification (double-T7). Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis. In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p. The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR. Both LM-PCR and double-T7 identified strongly bound genomic regions, but the double-T7 method increased sensitivity and specificity to allow detection of weaker binding sites.

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