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Publié par | technische_universitat_munchen |
Publié le | 01 janvier 2010 |
Nombre de lectures | 27 |
Langue | Deutsch |
Poids de l'ouvrage | 2 Mo |
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TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Physiologie
Impact of anabolics on miRNA and mRNA abundance in steroid target organs
Christiane Becker
Vollständiger Abdruck der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. K.-H. Engel
Prüfer der Dissertation: 1. Univ.-Prof. Dr. H.H.D. Meyer
2. Univ.-Prof. Dr. J.P. Geist
3. Priv.-Doz. Dr. M.W. Pfaffl
Die Dissertation wurde am 23.09.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 17.11.2010 angenommen.
Table of contents
Table of contents
Abbreviations………………………………… ......... ……………………………………….iii
Zusammenfassung…………………………………………………………………….....…vi
Abstract…… . ……………………………………………………………………………….viii
1 Introduction .......................................................................................................... 1
1.1 Anabolic steroids and their use in animal husbandry ................................................... 1
1.2 Transcriptomics as a potential method to detect biomarkers for anabolic treatment .... 2
1.3 miRNAs as a promising new target in gene expression analysis for biomarker
detection ....................................................................................................................... 4
1.4 Importance of RNA quality control in gene expression analysis ................................... 7
1.5 Aim of the thesis ........................................................................................................... 9
2 Material and methods..........................................................................................11
2.1 Collection of biological sample material ...................................................................... 11
2.1.1 Animal studies .................................................................................................. 11
2.1.2 Tissue and blood collection for methodological investigations ......................... 12
2.2 Purification of ribonucleic acids (RNA)........................................................................ 13
2.3 RNA degradation study ............................................................................................... 15
2.4 Analysis of total RNA integrity and small RNA quantification ..................................... 15
2.5 Target gene selection ................................................................................................. 16
2.6 Primer design .............................................................................................................. 17
2.7 cDNA synthesis .......................................................................................................... 22
2.7.1 cDNA synthesis for mRNA expression analysis ............................................... 22
2.7.2 esis for miRNA expression profiling using PCR arrays .................. 22
2.7.3 cDNA synthesis for miRNA expression analysis using single assay PCR ....... 22
2.8 Quantitative PCR (qPCR) ........................................................................................... 23
2.8.1 mRNA expression analysis ............................................................................... 23
2.8.2 miRNA expression analysis .............................................................................. 23
2.8.2.1 miRNA expression profiling using PCR arrays .............................................. 23
2.8.2.2 lysis using single assay PCR ................................... 24
2.9 Evaluation of gene expression data ............................................................................ 25
i
Table of contents
3 Results and discussion .......................................................................................27
3.1 mRNA studies ............................................................................................................. 27
3.1.1 Impact of trenbolone acetate plus estradiol-17 β on liver in Nguni heifers ........ 27
3.1.1.1 Plasma progesterone levels .......................................................................... 27
3.1.1.2 RNA Integrity ................................................................................................. 28
3.1.1.3 Gene expression analysis ............................................................................. 28
3.1.2 Influence of anabolic combinations of androgens plus estrogens on
reproductive tissues in post-pubertal Nguni heifers and pre-pubertal Holstein
Friesian calves .................................................................................................. 33
3.1.2.1 RNA integrity 33
3.1.2.2 Gene expression results from post-pubertal Nguni heifers and pre-pubertal
female Holstein Friesian calves ...................................................................... 33
3.2 miRNA studies ............................................................................................................ 43
3.2.1 Validation of miRNA analysis ............................................................................ 43
3.2.1.1 miRNA extraction .......................................................................................... 43
3.2.1.2 Verification of the Small RNA assay on the Agilent 2100 Bioanalyzer .......... 46
3.2.1.3 Impact of total RNA integrity on small RNA quantification and gene
expression analysis ......................................................................................... 47
3.2.2 Changes in the miRNA expression profile under the influence of TBA plus
E2 in bovine liver .............................................................................................. 56
4 Conclusions and perspectives ............................................................................63
5 References ......................................................................................................... 67
Acknowledgements………………………………………………………………………...75
List of scientific communications………………………………………………………….76
Curriculum vitae…………………………………………………………………………….79
Appendix…………………………………………………………………………………….80
ii
Abbreviations
Abbreviations
ACTB Actin β Ago argonaute protein
Alk anaplastic lymphoma ANGPT angiopoietin
receptor tyrosine kinase AP-1 activator protein 1
APO apolipoprotein AR androgen receptor
bcl-2 B-cell CLL/lymphoma 2 bcl-xl B-cell leukemia/lymphoma
BMP bone morphogenetic x
protein BMPR bone morphogenetic
bp base pair protein receptor
c-fos c-fos transcription factor c-jun c-jun transcription factor
c-kit v-kit Hardy-Zuckerman 4 c-myc myelocytomatosis cellular
feline sarcoma viral oncogen homolog
oncogen homolog CASP caspase
CAST calpastatin CEBP CCAAT/enhancer binding
CL corpus luteum protein
COX cyclooxygenase Cq quantitative cycle
CTSB cathepsin B CTSL cathepsin L
CV coefficient of variation CYL cyclin
CYP cytochrom P450 DEGMBE diethylen glycol
DMSO dimethylsulfoxid monobuthylether
(c)DNA (complementary) dNTP desoxyribonucleosid-
desoxyribonucleic acid triphosphat
ds double stranded E2 estradiol-17 β
EIA enzyme immunoassay ER estrogen receptor
EU European Union
iii
Abbreviations
FAS TNF receptor superfamily FASL TNF receptor superfamily
member 6 member 6 ligand
FDFT farnesyldiphosphat- FGF fibroblast growth factor
farnesyltransferase FLK-1 kinase insert domain
FLT-1 fms-related tyrosine kinase FSHR follicle stimulating hormone
receptor receptor
GDF growth differentiation factor GHR growth hormone receptor
GR glucocorticoid