Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study
<p>Abstract</p> <p>Background</p> <p>Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two exp...
| Main Authors: | , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2004-04-01
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| Series: | BMC Genomics |
| Online Access: | http://www.biomedcentral.com/1471-2164/5/26 |
| _version_ | 1811244598383607808 |
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| author | Wright Paul S Chen Yangde Zhang Xin Long Fan Cao Hui Diao Rong Lin Jieyi Ge Nanxiang DeLoughery Craig Liu Hong Epstein Charles Cao Wuxiong Busch Steve Wenck Michelle Wong Karen Saltzman Alan G Tang Zhihua Liu Li Zilberstein Asher |
| author_facet | Wright Paul S Chen Yangde Zhang Xin Long Fan Cao Hui Diao Rong Lin Jieyi Ge Nanxiang DeLoughery Craig Liu Hong Epstein Charles Cao Wuxiong Busch Steve Wenck Michelle Wong Karen Saltzman Alan G Tang Zhihua Liu Li Zilberstein Asher |
| author_sort | Wright Paul S |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip<sup>® </sup>technology. In this "case study" we explored the transcriptional pattern changes during the <it>in vitro </it>differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology.</p> <p>Results</p> <p>The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results.</p> <p>Conclusions</p> <p>This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.</p> |
| first_indexed | 2024-04-12T14:27:59Z |
| format | Article |
| id | doaj.art-d411f9aa97474bb0bc84bee25f1db141 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-12T14:27:59Z |
| publishDate | 2004-04-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-d411f9aa97474bb0bc84bee25f1db1412022-12-22T03:29:24ZengBMCBMC Genomics1471-21642004-04-01512610.1186/1471-2164-5-26Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case studyWright Paul SChen YangdeZhang XinLong FanCao HuiDiao RongLin JieyiGe NanxiangDeLoughery CraigLiu HongEpstein CharlesCao WuxiongBusch SteveWenck MichelleWong KarenSaltzman Alan GTang ZhihuaLiu LiZilberstein Asher<p>Abstract</p> <p>Background</p> <p>Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip<sup>® </sup>technology. In this "case study" we explored the transcriptional pattern changes during the <it>in vitro </it>differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology.</p> <p>Results</p> <p>The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results.</p> <p>Conclusions</p> <p>This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.</p>http://www.biomedcentral.com/1471-2164/5/26 |
| spellingShingle | Wright Paul S Chen Yangde Zhang Xin Long Fan Cao Hui Diao Rong Lin Jieyi Ge Nanxiang DeLoughery Craig Liu Hong Epstein Charles Cao Wuxiong Busch Steve Wenck Michelle Wong Karen Saltzman Alan G Tang Zhihua Liu Li Zilberstein Asher Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study BMC Genomics |
| title | Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study |
| title_full | Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study |
| title_fullStr | Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study |
| title_full_unstemmed | Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study |
| title_short | Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study |
| title_sort | comparing gene discovery from affymetrix genechip microarrays and clontech pcr select cdna subtraction a case study |
| url | http://www.biomedcentral.com/1471-2164/5/26 |
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