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Comparative analysis of a 3' end tag PCR and a linear RNA amplification approach for microarray analysis
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0003-3811-5439
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0002-4657-8532
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0003-4313-1601
2007 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 127, no 4, 638-646 p.Article in journal (Refereed) Published
Abstract [en]

 Background: Various types of amplification techniques have been developed in order to enable microarray gene expression analysis when the amount of starting material is limited. The two main strategies are linear amplification, using in vitro transcription, and exponential amplification, based on PCR. We have evaluated the performance of a linear and an in-house developed exponential amplification protocol that relies on 3' end tag sequences. We used 100 ng total RNA as starting material for amplification and compared the results with data from hybridizations with unamplified mRNA and total RNA.

Results: Preservation of expression ratios after amplification was examined comparing 1092 ratios obtained with amplification protocols to those obtained with standard labelling of mRNA. The Pearson correlations were 0.61 and 0.84, respectively, for the two linear amplification replicates and 0.76 and 0.80 for the two exponential amplification replicates. The correlations between repeated amplifications was 0.82 with the exponential method and 0.63 with the linear, indicating a better reproducibility with the PCR-based approach.

Conclusion: Both amplification methods generated results in agreement with unamplified material. In this study, the PCR-based method was more reproducible than in vitro transcription amplification. Advantages with the in-house developed method are the lower cost since it is non-commercial and that the PCR generated product offers compatibility with both sense and antisense arrays.

Place, publisher, year, edition, pages
2007. Vol. 127, no 4, 638-646 p.
Keyword [en]
microarray, linear amplification, exponential amplification, RNA amplification, in vitro transcription, PCR
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-6652DOI: 10.1016/j.jbiotec.2006.08.016ISI: 000243704600008Scopus ID: 2-s2.0-33845759906OAI: oai:DiVA.org:kth-6652DiVA: diva2:11422
Note
QC 20100903Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Microarray Based Gene Expression Analysis in Cancer Research
Open this publication in new window or tab >>Microarray Based Gene Expression Analysis in Cancer Research
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Biotechnological inventions during the 20th century have resulted in a wide range of approaches for explorations in the functional genomics field. Microarray technology is one of the recent advances which have provided us with snapshots of which genes are expressed in cells of various tissues and diseases. Methods to obtain reliable microarray data are continuously being developed and improved to meet the demands of biological researchers.

In this thesis microarrays have been used to investigate gene expression patterns in cancer research. Four studies in three different areas were carried out covering adrenocortical tumors, p53 target genes and a comparison of RNA amplification methods.

Adrenocortical tumours are among the most common tumours with an incidence of 7-9%. Malignancy of these tumors is rare. Distinction between malignant and benign tumours is often difficult to establish which makes an improvement of diagnostic approaches important. To elucidate biological processes in adrenocortical tumour development and to examine if there is a molecular signature associated with malignancy, microarray analysis was performed on 29 adrenocortical tumors and four normal specimens. It was possible to classify malignant and benign samples based on the entire expression profile. A number of potential biomarkers was identified which will be further evaluated.

P53 is a gene which is mutated in 50% of all cancers. Functional p53 is a transcription factor which is activated upon cellular stress and DNA damage. Target genes are mainly involved in cell cycle arrest and apoptosis. In solid tumors cells are stressed by hypoxia. To examine which target genes p53 activate under hypoxic conditions a microarray study of the cell lines HCT116p53+/+ and HCT116p53-/- was performed. A set of novel potential p53 target genes was identified while many known target genes were found to be not transcriptionally activated during hypoxia. Follow up which was focused on how p53 affected hypoxia induced apoptosis showed that the death receptor Fas was critical.

When small amounts of tissue are available, amplification of the transcript population is necessary for microarray analysis. A new strategy for amplification based on PCR was evaluated and compared to a commercial in vitro transcription protocol. Both protocols produced reliable results. Advantages with the PCR based method are a lower cost and a high flexibility due to compatibility with both sense and antisense strand microarrays.

Keywords: adrenocortical tumour, apoptosis, cancer, classification, gene expression, microarray, p53, RNA amplification

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 76 p.
Keyword
adrenocortical tumour, apoptosis, cancer, classification, gene expression, microarray, p53, RNA amplification
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4244 (URN)91-7178-542-6 (ISBN)978-91-7178-542-8 (ISBN)
Public defence
2006-12-22, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100907Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2010-09-07Bibliographically approved

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