High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis
Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was ap...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2004-11-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/04375RR02 |
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| author | Noëlani van den Berg Bridget G. Crampton Ingo Hein Paul R.J. Birch Dave K. Berger |
| author_facet | Noëlani van den Berg Bridget G. Crampton Ingo Hein Paul R.J. Birch Dave K. Berger |
| author_sort | Noëlani van den Berg |
| collection | DOAJ |
| description | Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis. |
| format | Article |
| id | doaj-art-b5b65a7bcb444984804e87c20b67c39c |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2004-11-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-b5b65a7bcb444984804e87c20b67c39c2025-08-20T02:25:36ZengTaylor & Francis GroupBioTechniques0736-62051940-98182004-11-0137581882410.2144/04375RR02High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysisNoëlani van den Berg0Bridget G. Crampton1Ingo Hein2Paul R.J. Birch3Dave K. Berger41University of Pretoria, Pretoria1University of Pretoria, Pretoria4Scottish Crop Research Institute, Invergowrie, Dundee, UK4Scottish Crop Research Institute, Invergowrie, Dundee, UK1University of Pretoria, PretoriaEfficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.https://www.future-science.com/doi/10.2144/04375RR02 |
| spellingShingle | Noëlani van den Berg Bridget G. Crampton Ingo Hein Paul R.J. Birch Dave K. Berger High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis BioTechniques |
| title | High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis |
| title_full | High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis |
| title_fullStr | High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis |
| title_full_unstemmed | High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis |
| title_short | High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis |
| title_sort | high throughput screening of suppression subtractive hybridization cdna libraries using dna microarray analysis |
| url | https://www.future-science.com/doi/10.2144/04375RR02 |
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