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Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach.

Ohnesorg T, Eggers S, Leonhard WN, Sinclair AH, White SJ - BMC Genomics (2009)

Bottom Line: We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment.Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC, Australia.

ABSTRACT

Background: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.

Results: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.

Conclusion: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

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MLPA on DNA from HeLa cells. A) Representative MLPA peak patterns obtained from nuclei isolated from HeLa cells and subsequent treatment with 0, 0.5 and 2 units of DNaseI. Red peaks represent size standards, blue peaks the signals from the probes after PCR amplification. Black numbers show probes designed to bind to non-sensitive regions, blue numbers to sensitive regions for HeLa cells as published by the ENCODE consortium. B) Analysis of experiment shown in 3A. Data were obtained from two independent experiments and two technical replicates (n = 4). Results shown as mean ± SD. C) Analysis of data derived from DNaseI treatment of naked DNA isolated from HeLa cells. In this case DNaseI digestion was with either 0.25 units or 1 unit DNaseI for 1 minute on ice, as this DNA was far more susceptible to degradation than DNA in intact nuclei. Probes are grouped into sensitive and non-sensitive, and then ordered according to their length. N: non-sensitive, S: sensitive for HeLa cells as published by the ENCODE consortium.
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Figure 3: MLPA on DNA from HeLa cells. A) Representative MLPA peak patterns obtained from nuclei isolated from HeLa cells and subsequent treatment with 0, 0.5 and 2 units of DNaseI. Red peaks represent size standards, blue peaks the signals from the probes after PCR amplification. Black numbers show probes designed to bind to non-sensitive regions, blue numbers to sensitive regions for HeLa cells as published by the ENCODE consortium. B) Analysis of experiment shown in 3A. Data were obtained from two independent experiments and two technical replicates (n = 4). Results shown as mean ± SD. C) Analysis of data derived from DNaseI treatment of naked DNA isolated from HeLa cells. In this case DNaseI digestion was with either 0.25 units or 1 unit DNaseI for 1 minute on ice, as this DNA was far more susceptible to degradation than DNA in intact nuclei. Probes are grouped into sensitive and non-sensitive, and then ordered according to their length. N: non-sensitive, S: sensitive for HeLa cells as published by the ENCODE consortium.

Mentions: We next used the same probe mix to analyse the DNaseI hypersensitivity of DNA within intact nuclei of HeLa cells. Figure 3A shows typical peak patterns obtained using nuclei aliquots digested with increasing amounts of DNaseI. Several peaks show significantly decreased peak height with increased DNaseI concentration, while others remain virtually unchanged. Nine of 11 probes targeting previously described DNaseI hypersensitive sites in HeLa cells show a clear decrease in normalized peak heights (defined as < 75% of the equivalent peak in undigested DNA), whereas all nine probes targeting non-sensitive loci show no significant decrease (figure 3B). These results were highly reproducible, with the technical and biological replicates giving an r2 > 0.9.


Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach.

Ohnesorg T, Eggers S, Leonhard WN, Sinclair AH, White SJ - BMC Genomics (2009)

MLPA on DNA from HeLa cells. A) Representative MLPA peak patterns obtained from nuclei isolated from HeLa cells and subsequent treatment with 0, 0.5 and 2 units of DNaseI. Red peaks represent size standards, blue peaks the signals from the probes after PCR amplification. Black numbers show probes designed to bind to non-sensitive regions, blue numbers to sensitive regions for HeLa cells as published by the ENCODE consortium. B) Analysis of experiment shown in 3A. Data were obtained from two independent experiments and two technical replicates (n = 4). Results shown as mean ± SD. C) Analysis of data derived from DNaseI treatment of naked DNA isolated from HeLa cells. In this case DNaseI digestion was with either 0.25 units or 1 unit DNaseI for 1 minute on ice, as this DNA was far more susceptible to degradation than DNA in intact nuclei. Probes are grouped into sensitive and non-sensitive, and then ordered according to their length. N: non-sensitive, S: sensitive for HeLa cells as published by the ENCODE consortium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748097&req=5

Figure 3: MLPA on DNA from HeLa cells. A) Representative MLPA peak patterns obtained from nuclei isolated from HeLa cells and subsequent treatment with 0, 0.5 and 2 units of DNaseI. Red peaks represent size standards, blue peaks the signals from the probes after PCR amplification. Black numbers show probes designed to bind to non-sensitive regions, blue numbers to sensitive regions for HeLa cells as published by the ENCODE consortium. B) Analysis of experiment shown in 3A. Data were obtained from two independent experiments and two technical replicates (n = 4). Results shown as mean ± SD. C) Analysis of data derived from DNaseI treatment of naked DNA isolated from HeLa cells. In this case DNaseI digestion was with either 0.25 units or 1 unit DNaseI for 1 minute on ice, as this DNA was far more susceptible to degradation than DNA in intact nuclei. Probes are grouped into sensitive and non-sensitive, and then ordered according to their length. N: non-sensitive, S: sensitive for HeLa cells as published by the ENCODE consortium.
Mentions: We next used the same probe mix to analyse the DNaseI hypersensitivity of DNA within intact nuclei of HeLa cells. Figure 3A shows typical peak patterns obtained using nuclei aliquots digested with increasing amounts of DNaseI. Several peaks show significantly decreased peak height with increased DNaseI concentration, while others remain virtually unchanged. Nine of 11 probes targeting previously described DNaseI hypersensitive sites in HeLa cells show a clear decrease in normalized peak heights (defined as < 75% of the equivalent peak in undigested DNA), whereas all nine probes targeting non-sensitive loci show no significant decrease (figure 3B). These results were highly reproducible, with the technical and biological replicates giving an r2 > 0.9.

Bottom Line: We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment.Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC, Australia.

ABSTRACT

Background: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.

Results: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.

Conclusion: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

Show MeSH
Related in: MedlinePlus