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Microarray analysis using disiloxyl 70mer oligonucleotides.

Björklund MG, Natanaelsson C, Karlström AE, Hao Y, Lundeberg J - Nucleic Acids Res. (2008)

Bottom Line: Technical knowledge regarding solid phase microarrays has also increased, and the results acquired today are more reliable than those obtained just a few years ago.We demonstrate that when the features on microarrays consist of full-length probes the signal intensity is significantly increased.The overall increase in intensity enables the hybridization stringency to be increased, and thus enhance the robustness of the results.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Department of Gene Technology, KTH, Royal Institute of Technology, AlbaNova University Center, SE-10691 Stockholm, Sweden.

ABSTRACT
DNA microarray technology has evolved dramatically in recent years, and is now a common tool in researchers' portfolios. The scope of the technique has expanded from small-scale studies to extensive studies such as classification of disease states. Technical knowledge regarding solid phase microarrays has also increased, and the results acquired today are more reliable than those obtained just a few years ago. Nevertheless, there are various aspects of microarray analysis that could be improved. In this article we show that the proportions of full-length probes used significantly affects the results of global analyses of transcriptomes. In particular, measurements of transcripts in low abundance are more sensitive to truncated probes, which generally increase the degree of cross hybridization and loss of specific signals. In order to improve microarray analysis, we here introduce a disiloxyl purification step, which ensures that all the probes on the microarray are at full length. We demonstrate that when the features on microarrays consist of full-length probes the signal intensity is significantly increased. The overall increase in intensity enables the hybridization stringency to be increased, and thus enhance the robustness of the results.

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(A) Distribution of A-values (the products of the intensities) for the hybridizations at 42°C and 50°C obtained with both types of probes using the amplified RNA data set. The distributions show a slight shift depending on probe type. (B) Box plot of hybridization data (50°C) for each of the 24 sequences using the amplified RNA data. Each box contains data across the different concentrations for each sequence in triplicate. The green and red boxes in each column represent data pertaining to the disiloxyl- and conventionally purified probes, respectively. (C) Distribution of A-values using the non-amplified RNA data set. (D) Box plot of hybridization data using the non-amplified RNA data set.
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Figure 3: (A) Distribution of A-values (the products of the intensities) for the hybridizations at 42°C and 50°C obtained with both types of probes using the amplified RNA data set. The distributions show a slight shift depending on probe type. (B) Box plot of hybridization data (50°C) for each of the 24 sequences using the amplified RNA data. Each box contains data across the different concentrations for each sequence in triplicate. The green and red boxes in each column represent data pertaining to the disiloxyl- and conventionally purified probes, respectively. (C) Distribution of A-values using the non-amplified RNA data set. (D) Box plot of hybridization data using the non-amplified RNA data set.

Mentions: First the hybridization accuracy and efficiency of the two sets of probes was investigated using the self/self hybridization data. For this purpose, the raw intensity data were transformed to log2-values using the product from the two channels (Cy3 and Cy5). The intensities were calculated for both the 42°C and the 50°C hybridizations. Initially, the differences between conventional probes and disiloxyl purified probes were investigated using ratio versus total intensity plots (MA plots). The MA plots are depicted in Figure 2, and the total intensity (A-value) distributions are shown in Figure 3A. Differences in A-values can be seen in the MA-plots so a t-test was carried out to compare the intensity distributions The resulting P-values for the 42°C and 50°C hybridizations were 1.053e-06 and 2.2e-16, respectively, indicating that there were significant differences between the intensities of the signals obtained with both sets of probes at the two different temperatures. The mean A-values (total intensity) for the two different probe types were 9.55 for the disiloxyl-purified probes and 8.93 for the conventional probes at 42°C. At 50°C the mean intensities were 9.41 and 8.40 for the disiloxyl-purified probes and the conventional probes, respectively, showing that the shift in intensities was larger for the conventional probes.Figure 2.


Microarray analysis using disiloxyl 70mer oligonucleotides.

Björklund MG, Natanaelsson C, Karlström AE, Hao Y, Lundeberg J - Nucleic Acids Res. (2008)

(A) Distribution of A-values (the products of the intensities) for the hybridizations at 42°C and 50°C obtained with both types of probes using the amplified RNA data set. The distributions show a slight shift depending on probe type. (B) Box plot of hybridization data (50°C) for each of the 24 sequences using the amplified RNA data. Each box contains data across the different concentrations for each sequence in triplicate. The green and red boxes in each column represent data pertaining to the disiloxyl- and conventionally purified probes, respectively. (C) Distribution of A-values using the non-amplified RNA data set. (D) Box plot of hybridization data using the non-amplified RNA data set.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2275082&req=5

Figure 3: (A) Distribution of A-values (the products of the intensities) for the hybridizations at 42°C and 50°C obtained with both types of probes using the amplified RNA data set. The distributions show a slight shift depending on probe type. (B) Box plot of hybridization data (50°C) for each of the 24 sequences using the amplified RNA data. Each box contains data across the different concentrations for each sequence in triplicate. The green and red boxes in each column represent data pertaining to the disiloxyl- and conventionally purified probes, respectively. (C) Distribution of A-values using the non-amplified RNA data set. (D) Box plot of hybridization data using the non-amplified RNA data set.
Mentions: First the hybridization accuracy and efficiency of the two sets of probes was investigated using the self/self hybridization data. For this purpose, the raw intensity data were transformed to log2-values using the product from the two channels (Cy3 and Cy5). The intensities were calculated for both the 42°C and the 50°C hybridizations. Initially, the differences between conventional probes and disiloxyl purified probes were investigated using ratio versus total intensity plots (MA plots). The MA plots are depicted in Figure 2, and the total intensity (A-value) distributions are shown in Figure 3A. Differences in A-values can be seen in the MA-plots so a t-test was carried out to compare the intensity distributions The resulting P-values for the 42°C and 50°C hybridizations were 1.053e-06 and 2.2e-16, respectively, indicating that there were significant differences between the intensities of the signals obtained with both sets of probes at the two different temperatures. The mean A-values (total intensity) for the two different probe types were 9.55 for the disiloxyl-purified probes and 8.93 for the conventional probes at 42°C. At 50°C the mean intensities were 9.41 and 8.40 for the disiloxyl-purified probes and the conventional probes, respectively, showing that the shift in intensities was larger for the conventional probes.Figure 2.

Bottom Line: Technical knowledge regarding solid phase microarrays has also increased, and the results acquired today are more reliable than those obtained just a few years ago.We demonstrate that when the features on microarrays consist of full-length probes the signal intensity is significantly increased.The overall increase in intensity enables the hybridization stringency to be increased, and thus enhance the robustness of the results.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Department of Gene Technology, KTH, Royal Institute of Technology, AlbaNova University Center, SE-10691 Stockholm, Sweden.

ABSTRACT
DNA microarray technology has evolved dramatically in recent years, and is now a common tool in researchers' portfolios. The scope of the technique has expanded from small-scale studies to extensive studies such as classification of disease states. Technical knowledge regarding solid phase microarrays has also increased, and the results acquired today are more reliable than those obtained just a few years ago. Nevertheless, there are various aspects of microarray analysis that could be improved. In this article we show that the proportions of full-length probes used significantly affects the results of global analyses of transcriptomes. In particular, measurements of transcripts in low abundance are more sensitive to truncated probes, which generally increase the degree of cross hybridization and loss of specific signals. In order to improve microarray analysis, we here introduce a disiloxyl purification step, which ensures that all the probes on the microarray are at full length. We demonstrate that when the features on microarrays consist of full-length probes the signal intensity is significantly increased. The overall increase in intensity enables the hybridization stringency to be increased, and thus enhance the robustness of the results.

Show MeSH
Related in: MedlinePlus