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Development of the first oligonucleotide microarray for global gene expression profiling in guinea pigs: defining the transcription signature of infectious diseases.

Jain R, Dey B, Tyagi AK - BMC Genomics (2012)

Bottom Line: To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection.A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection.An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.

ABSTRACT

Background: The Guinea pig (Cavia porcellus) is one of the most extensively used animal models to study infectious diseases. However, despite its tremendous contribution towards understanding the establishment, progression and control of a number of diseases in general and tuberculosis in particular, the lack of fully annotated guinea pig genome sequence as well as appropriate molecular reagents has severely hampered detailed genetic and immunological analysis in this animal model.

Results: By employing the cross-species hybridization technique, we have developed an oligonucleotide microarray with 44,000 features assembled from different mammalian species, which to the best of our knowledge is the first attempt to employ microarray to study the global gene expression profile in guinea pigs. To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection. A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection.

Conclusion: We report the development of first comprehensive microarray for studying the global gene expression profile in guinea pigs and validation of its usefulness with tuberculosis as a case study. An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.

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Distribution of probes in 44 K GPOM. The figure depicts the % distribution of oligonucleotide probes present in the 44 K guinea pig oligonucleotide microarray. The 60mer oligonucleotide probes were designed based on several mammalian species including human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), rhesus monkey (Macaca mulatta), dog (Canis familiaris), horse (Equus caballus), cat (Felis catus), sheep (Ovis aries), pig (Sus scrofa), chimpanzee (Pan troglodyte), chinchilla (Chinchilla lanigera), gray-tailed opossum (Monodelphis domestica), cattle (Bos taurus) and guinea pig (Cavia porcellus). The % representation of a particular species is calculated with respect to the total number of probes in the array. The figure does not show some of the mammalian species separately for which, the % representation is < 1% and are collectively labelled as – others. Number wise distribution of probes from all the species is given in Table 1.
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Figure 1: Distribution of probes in 44 K GPOM. The figure depicts the % distribution of oligonucleotide probes present in the 44 K guinea pig oligonucleotide microarray. The 60mer oligonucleotide probes were designed based on several mammalian species including human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), rhesus monkey (Macaca mulatta), dog (Canis familiaris), horse (Equus caballus), cat (Felis catus), sheep (Ovis aries), pig (Sus scrofa), chimpanzee (Pan troglodyte), chinchilla (Chinchilla lanigera), gray-tailed opossum (Monodelphis domestica), cattle (Bos taurus) and guinea pig (Cavia porcellus). The % representation of a particular species is calculated with respect to the total number of probes in the array. The figure does not show some of the mammalian species separately for which, the % representation is < 1% and are collectively labelled as – others. Number wise distribution of probes from all the species is given in Table 1.

Mentions: For gene expression profiling of species that lack genome sequence and/or representative microarray platforms, cross-species hybridization based microarray has conventionally been used. Since, fully annotated guinea pig genome sequence is not available, we employed cross-species hybridization technology to develop a 44 K microarray platform to study gene expression profile in guinea pigs. As described in the Additional file1 and Supporting Table S1, initially a 244 K microarray was designed to contain 60 mer oligonucleotide probes from multiple mammalian species (human, mouse, rat, guinea pig, rhesus monkey, dog, horse, cat, sheep, pig, chimpanzee, chinchilla, gray-tailed opossum and cattle) based on all the probe sequences available from Agilent Catalogue arrays and NCBI mRNA sequences. Especially, the array included 1132 probes based on annotated gene sequences of guinea pig and 92,815 probes corresponding to guinea pig ESTs. The 244 K array was then hybridized with Cy3 labeled cRNA produced from pooled RNA obtained from various guinea pig tissues (lung, liver, spleen, brain, muscle, kidney and bone marrow) and Cy5 labeled genomic DNA isolated from guinea pig spleen tissue. Following hybridization, the array was scanned and features were extracted. The filtration criteria during the probe selection, while developing microarray by cross-species hybridization technology on Agilent platform, are based on comparison of specific signal intensity viz. the background signal intensity. Probes exhibiting significantly higher signal intensity (p < 0.05), at least 2 fold higher as compared to the background are selected for array development. Based on this criterion, a total of 20,023 out of 62,560 probes representing different mammalian genes were selected from the 244 K array. Similarly, a total of 9,823 out of 92,815 probes were selected for ESTs. However, irrespective of the intensities, all the 1,132 probes for guinea pig were included. Further, an additional of 12,825 best probes out of 19,975 newly added guinea pig EST’s from NCBI database were added to the 44 K array (Table1). Thus, the final design of the guinea pig 44 K microarray comprised of a total number of 45,220 features including 29,846 valid features from different mammalian species (Figure1), 1,132 probes for guinea pig transcripts and 12,825 probes for guinea pig ESTs, 1,264 Agilent positive controls and 153 Agilent negative controls. Agilent positive and negative controls are standard set of probes employed by the Agilent microarray platform for mammalian microarray studies. The negative control probes are intended to have no hybridization and these are used by feature extraction software for background determination. The positive controls are used to have predictable signals, which are used for monitoring the microarray linearity, sensitivity and accuracy. Based on the above-mentioned method for probe selection, many genes are represented by multiple but unique probes derived from different mammalian species. Use of multiple unique probes per transcript in general increases the confidence of microarray result. The averaging of the signals from multiple probes provides improved statistical confidence, reducing the impact of inconsistent probe behavior and improving the signal to noise ratio compared to the platforms that offer fewer probes per gene. For biological interpretation, homolog Gene ontology annotation was also obtained for all the probes by blast-based homology to reference sequence database of human, mouse and rat for which validated methods for biological pathway analysis are available.


Development of the first oligonucleotide microarray for global gene expression profiling in guinea pigs: defining the transcription signature of infectious diseases.

Jain R, Dey B, Tyagi AK - BMC Genomics (2012)

Distribution of probes in 44 K GPOM. The figure depicts the % distribution of oligonucleotide probes present in the 44 K guinea pig oligonucleotide microarray. The 60mer oligonucleotide probes were designed based on several mammalian species including human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), rhesus monkey (Macaca mulatta), dog (Canis familiaris), horse (Equus caballus), cat (Felis catus), sheep (Ovis aries), pig (Sus scrofa), chimpanzee (Pan troglodyte), chinchilla (Chinchilla lanigera), gray-tailed opossum (Monodelphis domestica), cattle (Bos taurus) and guinea pig (Cavia porcellus). The % representation of a particular species is calculated with respect to the total number of probes in the array. The figure does not show some of the mammalian species separately for which, the % representation is < 1% and are collectively labelled as – others. Number wise distribution of probes from all the species is given in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475082&req=5

Figure 1: Distribution of probes in 44 K GPOM. The figure depicts the % distribution of oligonucleotide probes present in the 44 K guinea pig oligonucleotide microarray. The 60mer oligonucleotide probes were designed based on several mammalian species including human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), rhesus monkey (Macaca mulatta), dog (Canis familiaris), horse (Equus caballus), cat (Felis catus), sheep (Ovis aries), pig (Sus scrofa), chimpanzee (Pan troglodyte), chinchilla (Chinchilla lanigera), gray-tailed opossum (Monodelphis domestica), cattle (Bos taurus) and guinea pig (Cavia porcellus). The % representation of a particular species is calculated with respect to the total number of probes in the array. The figure does not show some of the mammalian species separately for which, the % representation is < 1% and are collectively labelled as – others. Number wise distribution of probes from all the species is given in Table 1.
Mentions: For gene expression profiling of species that lack genome sequence and/or representative microarray platforms, cross-species hybridization based microarray has conventionally been used. Since, fully annotated guinea pig genome sequence is not available, we employed cross-species hybridization technology to develop a 44 K microarray platform to study gene expression profile in guinea pigs. As described in the Additional file1 and Supporting Table S1, initially a 244 K microarray was designed to contain 60 mer oligonucleotide probes from multiple mammalian species (human, mouse, rat, guinea pig, rhesus monkey, dog, horse, cat, sheep, pig, chimpanzee, chinchilla, gray-tailed opossum and cattle) based on all the probe sequences available from Agilent Catalogue arrays and NCBI mRNA sequences. Especially, the array included 1132 probes based on annotated gene sequences of guinea pig and 92,815 probes corresponding to guinea pig ESTs. The 244 K array was then hybridized with Cy3 labeled cRNA produced from pooled RNA obtained from various guinea pig tissues (lung, liver, spleen, brain, muscle, kidney and bone marrow) and Cy5 labeled genomic DNA isolated from guinea pig spleen tissue. Following hybridization, the array was scanned and features were extracted. The filtration criteria during the probe selection, while developing microarray by cross-species hybridization technology on Agilent platform, are based on comparison of specific signal intensity viz. the background signal intensity. Probes exhibiting significantly higher signal intensity (p < 0.05), at least 2 fold higher as compared to the background are selected for array development. Based on this criterion, a total of 20,023 out of 62,560 probes representing different mammalian genes were selected from the 244 K array. Similarly, a total of 9,823 out of 92,815 probes were selected for ESTs. However, irrespective of the intensities, all the 1,132 probes for guinea pig were included. Further, an additional of 12,825 best probes out of 19,975 newly added guinea pig EST’s from NCBI database were added to the 44 K array (Table1). Thus, the final design of the guinea pig 44 K microarray comprised of a total number of 45,220 features including 29,846 valid features from different mammalian species (Figure1), 1,132 probes for guinea pig transcripts and 12,825 probes for guinea pig ESTs, 1,264 Agilent positive controls and 153 Agilent negative controls. Agilent positive and negative controls are standard set of probes employed by the Agilent microarray platform for mammalian microarray studies. The negative control probes are intended to have no hybridization and these are used by feature extraction software for background determination. The positive controls are used to have predictable signals, which are used for monitoring the microarray linearity, sensitivity and accuracy. Based on the above-mentioned method for probe selection, many genes are represented by multiple but unique probes derived from different mammalian species. Use of multiple unique probes per transcript in general increases the confidence of microarray result. The averaging of the signals from multiple probes provides improved statistical confidence, reducing the impact of inconsistent probe behavior and improving the signal to noise ratio compared to the platforms that offer fewer probes per gene. For biological interpretation, homolog Gene ontology annotation was also obtained for all the probes by blast-based homology to reference sequence database of human, mouse and rat for which validated methods for biological pathway analysis are available.

Bottom Line: To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection.A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection.An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.

ABSTRACT

Background: The Guinea pig (Cavia porcellus) is one of the most extensively used animal models to study infectious diseases. However, despite its tremendous contribution towards understanding the establishment, progression and control of a number of diseases in general and tuberculosis in particular, the lack of fully annotated guinea pig genome sequence as well as appropriate molecular reagents has severely hampered detailed genetic and immunological analysis in this animal model.

Results: By employing the cross-species hybridization technique, we have developed an oligonucleotide microarray with 44,000 features assembled from different mammalian species, which to the best of our knowledge is the first attempt to employ microarray to study the global gene expression profile in guinea pigs. To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection. A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection.

Conclusion: We report the development of first comprehensive microarray for studying the global gene expression profile in guinea pigs and validation of its usefulness with tuberculosis as a case study. An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.

Show MeSH
Related in: MedlinePlus