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Transcript profiling of small tissue samples using microarray technology
KTH, School of Biotechnology (BIO).
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Through a number of biological, technological and computational achievements during the 20th century and the devoted work of hundreds of researchers the sequence of the human and other genomes are now available in public databases. The current challenge is to begin to understand the information encoded by the DNA sequence, to elucidate the functions of the proteins and RNA molecules encoded by the genes as well as how they are regulated. For this purpose new technologies within the area of functional genomics are being developed. Among those are powerful tools for gene expression analysis, such as microarrays, providing means to investigate when and where certain genes are used.

This thesis describes a method that was developed to enable gene expression analysis, on the transcriptome level, in small tissue samples. It relies on PCR amplification of the 3’-ends of cDNA (denoted 3’-end signature tags). PCR is a powerful technology for amplification of nucleic acids, but has not been used much for transcript profiling since it is generally considered to introduce biases, distorting the original relative transcript levels. The described method addresses this issue by generating uniformly sized representatives of the transcripts/cDNAs prior to amplification. This is achieved through sonication which, unlike restriction enzymes, does not require a specific recognition sequence and fragments each transcript randomly. The method was evaluated using cDNA microarrays, Affymetrix™ oligonucleotide arrays and real-time quantitative PCR. It was shown to perform well, yielding transcript profiles that correlate well to the original, unamplified material, as well as being highly reproducible.

The developed method was applied to stem cell biology. The variability in gene expression between different populations of cultured neural stem cells (neurospheres) was investigated. It was shown that neurospheres isolated from different animals or passaged to different degrees show large fluctuations in gene expression, while neurospheres isolated and cultured under identical conditions are more similar and suitable for gene expression analysis. A second study showed that withdrawing epidermal growth factor (EGF) from the culture medium when treating the cells with an agent of interest has profound effects on gene expression, something which should be taken into consideration in future neurosphere studies.

Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , 89 p.
Keyword [en]
Biology, Gene expression analysis, transcriptomics, microarray analysis, 3’-tag signature amplification, neural stem cells
Keyword [sv]
Biologi
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-158ISBN: 91-7283-989-9 (print)OAI: oai:DiVA.org:kth-158DiVA: diva2:7505
Public defence
2005-04-08, Kollegiesalen, KTH, Valhallavägen 79, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101006Available from: 2005-04-04 Created: 2005-04-04 Last updated: 2010-10-06Bibliographically approved
List of papers
1. cDNA microarray analysis of small tissue samples using a cDNA tag target amplification protocol
Open this publication in new window or tab >>cDNA microarray analysis of small tissue samples using a cDNA tag target amplification protocol
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2001 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 25, no 5, 585-591 p.Article in journal (Refereed) Published
Abstract [en]

Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50-200 mug of total RNA and 1-2 mug of mRNA is required for each hybridisation, which is equivalent to 50-100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub-mg amounts of plant tissue. Using 0.1 mug of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2-4 cell layers with a fresh weight of similar to 0.5 mg.

Keyword
PCR, cDNA-amplification, microarray, transcript profiling, phloem development, populus tremula x tremuloides
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-5016 (URN)10.1046/j.1365-313x.2001.00972.x (DOI)000168419300011 ()
Note
QC 20101006 NR 20140805Available from: 2005-04-04 Created: 2005-04-04 Last updated: 2017-12-05Bibliographically approved
2. Amplification of mRNA populations by a cDNA tag strategy
Open this publication in new window or tab >>Amplification of mRNA populations by a cDNA tag strategy
2004 (English)In: BioTechniques, ISSN 0736-6205, Vol. 36, no 2, 253-259 p.Article in journal (Refereed) Published
Abstract [en]

Here we describe an amplification method for global transcript analysis. The strategy relies on amplification of cDNA tags (signature tags) achieved by random fragmentation of the cDNAs to short tags of similar length, isolation of the 3' ends and then PCR amplification of the 3'-end signature tag population. This method minimizes biased amplification that may occur during parallel amplification of long and short templates. The amplified tags can be either cloned and sequenced or labeled and hybridized to DNA arrays to identify the expressed transcripts. To verify that the relative levels between transcripts in different mRNA/cDNA populations are maintained during the amplification protocol, we have used the Affymetrix oligonucleotide platform and real-time PCR.

Keyword
Amplification, Cloning, DNA, Real time systems
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-5017 (URN)000188882400011 ()2-s2.0-0842309819 (Scopus ID)
Note

QC 20100827

Available from: 2005-04-04 Created: 2005-04-04 Last updated: 2017-06-09Bibliographically approved
3. Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method
Open this publication in new window or tab >>Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method
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2005 (English)In: BMC neuroscience (Online), ISSN 1471-2202, Vol. 6, no 28, 13- p.Article in journal (Refereed) Published
Abstract [en]

Background: Neural stem cells ( NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression.

Results: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability.

Conclusions: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

Keyword
central-nervous-system, adult human brain, microarray data, differentiation, neurospheres, neurons, expression, generation, single
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6167 (URN)10.1186/1471-2202-6-28 (DOI)000229042300001 ()2-s2.0-26444571449 (Scopus ID)
Note
QC 20100927Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2010-10-06Bibliographically approved
4. Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation
Open this publication in new window or tab >>Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation
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2005 (English)In: BMC neuroscience (Online), ISSN 1471-2202, Vol. 6, 55- p.Article in journal (Refereed) Published
Abstract [en]

Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.

Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP.

Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.

Keyword
CENTRAL-NERVOUS-SYSTEM, ADULT-MOUSE BRAIN, PARKINSONS-DISEASE, MICROARRAY DATA, DENTATE GYRUS, DIFFERENTIATION, TRANSPLANTATION, AMPLIFICATION, PROGENITORS, NEURONS
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6168 (URN)10.1186/1471-2202-6-55 (DOI)000231880900001 ()2-s2.0-26444590557 (Scopus ID)
Note
QC 20100914Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2010-10-06Bibliographically approved

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