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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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Comparison of results obtained using different cell numbers. Correlation of results obtained from experiments with 5 × 104 (n = 1) and 2.5 × 105 (n = 4) HeLa cells per DNaseI treatment aliquot.
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Figure 5: Comparison of results obtained using different cell numbers. Correlation of results obtained from experiments with 5 × 104 (n = 1) and 2.5 × 105 (n = 4) HeLa cells per DNaseI treatment aliquot.

Mentions: We also tried to determine the minimum cell number that could be used with this approach. As we were able to obtain robust results and sufficient DNA for several replicates from aliquots containing nuclei from about 2.5 × 105 cells, we estimated the lower limit of required cells to be around 5 × 104 (theoretically ~300 ng DNA). To confirm this number, we used aliquots of 5 × 104 HeLa cells and treated them as described above and compared the results with those obtained from experiments using 2.5 × 105 cells per aliquot. Figure 5 shows the comparison of results from 5 × 104 and 2.5 × 105 cells (r2 = 0.97).


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)

Comparison of results obtained using different cell numbers. Correlation of results obtained from experiments with 5 × 104 (n = 1) and 2.5 × 105 (n = 4) HeLa cells per DNaseI treatment aliquot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Comparison of results obtained using different cell numbers. Correlation of results obtained from experiments with 5 × 104 (n = 1) and 2.5 × 105 (n = 4) HeLa cells per DNaseI treatment aliquot.
Mentions: We also tried to determine the minimum cell number that could be used with this approach. As we were able to obtain robust results and sufficient DNA for several replicates from aliquots containing nuclei from about 2.5 × 105 cells, we estimated the lower limit of required cells to be around 5 × 104 (theoretically ~300 ng DNA). To confirm this number, we used aliquots of 5 × 104 HeLa cells and treated them as described above and compared the results with those obtained from experiments using 2.5 × 105 cells per aliquot. Figure 5 shows the comparison of results from 5 × 104 and 2.5 × 105 cells (r2 = 0.97).

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