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Interpreting the human transcriptome
KTH, School of Biotechnology (BIO), Gene Technology. (Joakim Lundeberg)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed.

The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population.

Abstract [sv]

Människokroppen är uppbyggd av miljarder celler och nästan alla innehåller samma arvsmassa. Trots detta finns det många olika celler med olika funktioner vilket är en följd av vilken del av arvsmassan som cellerna använder, dvs vilka RNA-molekyler som finns i varje cell. Den snabba utvecklingen av sekvenseringstekniker har gjort det möjligt att studera när, var och hur varje RNA-molekyl är uttryckt och att få en djupare förståelse för hur människans celler fungerar.

Arbetet som presenteras i denna avhandling fokuserar på analys av RNA-molekyler i människans celler. I artikel I beskriver vi en automatiserad metod för att förbereda cellprov för RNA-sekvensering. Det automatiserade protokollet jämfördes med det manuella protokollet, och vi visade att det automatiserade protokollet överträffade det manuella när det gällde provkapacitet samtidigt som en höga reproducerbarheten behölls. I artikel II undersökte vi effekterna som RNA-molekyler från en del av cellen (cellkärnan) har på den totala mängden uttryckta RNA-molekyler. Vi jämförde RNA från hela cellen och från en del av cellen (cytoplasman) och visade att RNA-molekyler med långa och strukturerade 3'- och 5'-otranslaterade regioner och RNA-molekyler med långa proteinkodande sekvenser tenderade att hållas kvar i cellkärnan till en högre grad. Detta resulterade i en ökad komplexitet av RNA-molekylerna i hela cellen, medan vi i cytoplasma-fraktionen lättare kunde hitta de korta och svagt uttryckta RNA-molekyler. I Artikel III och IV studerar vi RNA-molekyler i människans skelettmuskler. I artikel III visar vi att andelen RNA-molekyler uttryckta i skelettmuskler är väldigt lika mellan muskler och mellan olika personer, men att ett stort antal RNA-molekyler var uttryckta i olika nivåer hos kvinnor och män. Artikel IV beskriver RNA-nivåer som svar på upprepade perioder av uthållighetsträning. Artikel V beskriver en metod för att studera ett fåtal utvalda RNA-molekyler. Vi valde RNA-molekyler vars uttryck är viktigt vid analys av bröstcancerceller, och optimerade metoden för analys av enskilda celler. Vi analyserade cancerceller från blodprov och använde metoden för att titta på RNA-nivåer i enskilda celler från en grupp av celler och visade på skillnader i RNA-nivåer inom gruppen.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. , 52 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:1
Keyword [en]
Transcriptome, RNA sequencing, high-throughput sequencing, gene expression profiling, multiplex amplification
National Category
Cell and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-158320ISBN: 978-91-7595-413-4 (print)OAI: oai:DiVA.org:kth-158320DiVA: diva2:776188
Public defence
2015-02-06, Air & Fire, Science for Life laboratory, Tomtebodavägen 23A, Solna, 10:29 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Cancer SocietyEU, FP7, Seventh Framework Programme, FP7‐ICT‐257743
Note

QC 20150115

Available from: 2015-01-15 Created: 2015-01-07 Last updated: 2015-01-15Bibliographically approved
List of papers
1. Scalable Transcriptome Preparation for Massive Parallel Sequencing
Open this publication in new window or tab >>Scalable Transcriptome Preparation for Massive Parallel Sequencing
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 7, e21910- p.Article in journal (Refereed) Published
Abstract [en]

Background: The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity. Methodology: In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation. Conclusion/Significance: The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-37159 (URN)10.1371/journal.pone.0021910 (DOI)000292655400026 ()2-s2.0-79960041380 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20110803Available from: 2011-08-03 Created: 2011-08-02 Last updated: 2017-12-08Bibliographically approved
2. Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs
Open this publication in new window or tab >>Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs
Show others...
2012 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 13, no 1, 574- p.Article in journal (Refereed) Published
Abstract [en]

Background: The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells (total RNA) were prepared from three human cell lines and sequenced using massive parallel sequencing. Results: For all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level. Conclusions: We conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs.

Keyword
Differential detection, Gene expression, Nuclear retention, miRNA regulation, RNA-Seq
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-107092 (URN)10.1186/1471-2164-13-574 (DOI)000311057300001 ()2-s2.0-84869174021 (Scopus ID)
Funder
Swedish Research CouncilEU, European Research Council, 257743 222913Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121206

Available from: 2012-12-06 Created: 2012-12-06 Last updated: 2017-12-07Bibliographically approved
3. The human skeletal muscle transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing
Open this publication in new window or tab >>The human skeletal muscle transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing
Show others...
2014 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 10, 4571-4581 p.Article in journal (Refereed) Published
Abstract [en]

Human skeletal muscle health is important for quality of life and several chronic diseases, including type II diabetes, heart disease, and cancer. Skeletal muscle is a tissue widely used to study mechanisms behind different diseases and adaptive effects of controlled interventions. For such mechanistic studies, knowledge about the gene expression profiles in different states is essential. Since the baseline transcriptome has not been analyzed systematically, the purpose of this study was to provide a deep reference profile of female and male skeletal muscle. RNA sequencing data were analyzed from a large set of 45 resting human muscle biopsies. We provide extensive information on the skeletal muscle transcriptome, including 5 previously unannotated protein-coding transcripts. Global transcriptional tissue homogeneity was strikingly high, within both a specific muscle and the contralateral leg. We identified >23,000 known isoforms and found >5000 isoforms that differ between the sexes. The female and male transcriptome was enriched for genes associated with oxidative metabolism and protein catabolic processes, respectively. The data demonstrate remarkably high tissue homogeneity and provide a deep and extensive baseline reference for the human skeletal muscle transcriptome, with regard to alternative splicing, novel transcripts, and sex differences in functional ontology.

Keyword
gene expression profiling, biopsy, isoform, splice variant, novel transcript
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-154744 (URN)10.1096/fj.14-255000 (DOI)000342222700032 ()2-s2.0-84907688003 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20141105

Available from: 2014-11-05 Created: 2014-10-27 Last updated: 2017-12-05Bibliographically approved
4. Long-­‐term endurance training induces global isoform changes in human skeletal muscle
Open this publication in new window or tab >>Long-­‐term endurance training induces global isoform changes in human skeletal muscle
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(English)Manuscript (preprint) (Other academic)
Keyword
Myocyte, exercise, cell memory, gene expression, alternative splicing, detraining, tapering, novel isoforms
Identifiers
urn:nbn:se:kth:diva-158931 (URN)
Note

QS 2015

Available from: 2015-01-15 Created: 2015-01-15 Last updated: 2015-01-15Bibliographically approved
5. Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring
Open this publication in new window or tab >>Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring
Show others...
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-158933 (URN)
Note

QS 2015

Available from: 2015-01-15 Created: 2015-01-15 Last updated: 2015-01-15Bibliographically approved

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