Impact of anabolics on miRNA and mRNA abundance in steroid target organs [Elektronische Ressource] / Christiane Becker
171 pages
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Impact of anabolics on miRNA and mRNA abundance in steroid target organs [Elektronische Ressource] / Christiane Becker

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171 pages
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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 ......................................................

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Publié par
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

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