Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Amplification of mRNA populations by a cDNA tag strategy
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-4657-8532
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4313-1601
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.

Place, publisher, year, edition, pages
2004. Vol. 36, no 2, 253-259 p.
Keyword [en]
Amplification, Cloning, DNA, Real time systems
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-5017ISI: 000188882400011Scopus ID: 2-s2.0-0842309819OAI: oai:DiVA.org:kth-5017DiVA: diva2:7504
Note

QC 20100827

Available from: 2005-04-04 Created: 2005-04-04 Last updated: 2017-06-09Bibliographically approved
In thesis
1. Transcript profiling of small tissue samples using microarray technology
Open this publication in new window or tab >>Transcript profiling of small tissue samples using microarray technology
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
Biology, Gene expression analysis, transcriptomics, microarray analysis, 3’-tag signature amplification, neural stem cells, Biologi
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-158 (URN)91-7283-989-9 (ISBN)
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

Open Access in DiVA

No full text

Scopus

Search in DiVA

By author/editor
Sievertzon, MariaAgaton, CharlottaNilsson, PeterLundeberg, Joakim
By organisation
Biotechnology
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 53 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf