Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR
Background: Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematical...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BioMed Central Ltd
2011
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| Online Access: | http://hdl.handle.net/1721.1/64665 https://orcid.org/0000-0001-6909-4568 |
| _version_ | 1811092854065332224 |
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| author | Zhou, Kang Zhou, Lihan Lim, Qing 'En Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Zhou, Kang Zhou, Lihan Lim, Qing 'En Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon |
| author_sort | Zhou, Kang |
| collection | MIT |
| description | Background: Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results: In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion: This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli. |
| first_indexed | 2024-09-23T15:30:35Z |
| format | Article |
| id | mit-1721.1/64665 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T15:30:35Z |
| publishDate | 2011 |
| publisher | BioMed Central Ltd |
| record_format | dspace |
| spelling | mit-1721.1/646652022-09-29T14:49:14Z Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR Zhou, Kang Zhou, Lihan Lim, Qing 'En Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon Massachusetts Institute of Technology. Department of Chemical Engineering Stephanopoulos, Gregory Background: Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results: In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion: This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli. Singapore-MIT Alliance 2011-06-23T17:03:13Z 2011-06-23T17:03:13Z 2011-04 2011-01 2011-06-07T15:10:50Z Article http://purl.org/eprint/type/JournalArticle 1471-2199 http://hdl.handle.net/1721.1/64665 BMC Molecular Biology. 2011 Apr 23;12(1):18 21513543 https://orcid.org/0000-0001-6909-4568 en http://dx.doi.org/10.1186/1471-2199-12-18 BMC Molecular Biology Creative Commons Attribution http://creativecommons.org/licenses/by/2.0 Zhou et al.; licensee BioMed Central Ltd. application/pdf BioMed Central Ltd BioMed Central Ltd |
| spellingShingle | Zhou, Kang Zhou, Lihan Lim, Qing 'En Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR |
| title | Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR |
| title_full | Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR |
| title_fullStr | Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR |
| title_full_unstemmed | Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR |
| title_short | Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR |
| title_sort | novel reference genes for quantifying transcriptional responses of escherichia coli to protein overexpression by quantitative pcr |
| url | http://hdl.handle.net/1721.1/64665 https://orcid.org/0000-0001-6909-4568 |
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