Quantitative single-cell RT-PCR analysis of the TRPC channel subunits expression patterns in cerebellar Purkinje neurons [Elektronische Ressource] / Elena Dragicevic

Dragicevic

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

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Friedrich-Schiedel Institut für Neurowissenschaften
Technische Universität München

Quantitative single-cell RT-PCR analysis of the
TRPC channel subunits expression patterns in
cerebellar Purkinje neurons

Elena Dragicevic

Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität
München zur Erlangung des akademischen Grades eines

Doctor of Philosophy (Ph.D.)

genehmigten Dissertation.

Vorsitzender: apl. Prof. Dr. Helmuth Adelsberger
Prüfer der Dissertation:
1. Univ.-Prof. Dr. Arthur Konnerth
2. Dr. Michael Schemann

Die Dissertation wurde am 20.12.2007 beim Studienausschuss des Ph.D. Studiengangs
Medical Life Science and Technology an der Fakultät für Medizin der Technischen
Universität München eingereicht und durch die Fakultät für Medizin am 31.03.2008
angenommen. Table of Contents

Table of Contents
............................................................................................................... I

Glossary ......................................................................................................................... IV

1.  Introduction ..................................................................................................... 1 
1.1  The TRPC channel family ................................................................................. 1 
1.2  TRPC channels in cerebellar Purkinje neurons ................................................ 7 
1.3  Quantitative single-cell RT-PCR ..................................................................... 10 
1.4  Experimental goals .......................................................................................... 12

2.  Materials and Methods 13 
2.1  Materials .......................................................................................................... 13 
2.1.1  Chemicals ....................................................................................................... 13 
2.1.2  Buffers and solutions ....................................................................................... 13 
2.1.3  Enzymes and enzyme buffers ......................................................................... 15 
2.1.4  Antibodies ....................................................................................................... 16 
2.1.5  Nucleotides and oligonucleotides .................................................................... 16 
2.1.6  Vectors ............................................................................................................ 16 
2.1.7  Dyes and markers ........................................................................................... 16 
2.1.8  Kits .................................................................................................................. 16 
2.1.9  Microorganisms ............................................................................................... 17 
2.1.10  Animals ........................................................................................................... 17 
2.2  Methods .......................................................................................................... 17 
2.2.1  Cloning ............................................................................................................ 17 
2.2.1.1  Ligation ............................................................................................................ 17 
2.2.1.2  Transformation of E.Coli bacterial strains with plasmid DNA .......................... 18 
2.2.2  Isolation and purification of DNA ..................................................................... 18 
2.2.2.1  Small scale DNA isolation (Mini-prep) ............................................................. 18 
2.2.2.2  Large scale DNA isolation (Maxi-prep) ........................................................... 19 
2.2.2.3  Long scale storage of clones .......................................................................... 20 
2.2.2.4  Isolation and purification of DNA fragments from agarose gels ...................... 20 
2.2.2.5  Purification of PCR products ........................................................................... 21 
2.2.3  Isolation and purification of RNA 22 
2.2.4  DNA electrophoresis ....................................................................................... 22 
2.2.4.1  DNA electrophoresis on agarose gels ............................................................. 22 
I
Table of Contents

2.2.4.1.1  Analytical gels ................................................................................................. 23 
2.2.4.1.2  Preparative gels .............................................................................................. 23 
2.2.4.2  DNA size markers ........................................................................................... 23 
2.2.5  RNA electrophoresis ....................................................................................... 23 
2.2.6  Quantification of DNA and RNA ...................................................................... 24 
2.2.7  DNA analysis with restriction endonucleases ................................................. 24 
2.2.8  Cell harvest ..................................................................................................... 24 
2.2.8.1  Preparation of glass capillaries for cell harvest ............................................... 24 
2.2.8.2  Preparation of cerebellar slices ....................................................................... 25 
2.2.8.3  Cell harvest procedure .................................................................................... 25 
2.2.9  Reverse Transcription (RT) ............................................................................. 26 
2.2.9.1 nscription of total RNA ................................................................. 26 
2.2.9.2  Reverse Tranof single-cell material .................................................. 27 
2.2.10  cDNA purification ............................................................................................ 28 
2.2.11  Polymerase Chain Reaction (PCR) 29 
2.2.11.1  Primer selection .............................................................................................. 29 
2.2.11.2  Nested PCR .................................................................................................... 30 
2.2.12  Real-time quantitative PCR ............................................................................. 32 
2.2.12.1  Basis of real-time quantitative PCR ................................................................ 32 
TM2.2.12.2  The LightCycler ............................................................................................ 33 
TM2.2.12.3  Quantification on the LightCycler .............................................................. 34 
2.2.12.4  Construction of high-resolution external standard curves ............................... 35 
2.2.12.5  Rapid-cycle, real-time PCR 36

3.  Results ........................................................................................................... 37 
3.1  Nested PCR .................................................................................................... 37 
3.2  Gene-specific primers selected for the real-time PCR .................................... 39 
3.3  Optimization of real-time PCR cycles .............................................................. 40 
3.4  Verification of the PCR products by sequencing ............................................. 40 
3.5  High-resolution external standard curves ........................................................ 41 
3.6  Verification of external standard curves efficiencies ....................................... 48 
3.7  Calculation of TRPC subunit copy numbers ................................................... 55 
3.8  Quantification of TRPC subunit expression in the brain .................................. 56 
3.9  Quantitative single-cell RT-PCR ..................................................................... 57 
3.9.1  Control experiments ........................................................................................ 59 
3.9.1.1  Cell harvest controls ........................................................................................ 59 
3.9.1.2  RT efficiency controls ...................................................................................... 61 
3.9.2  TRPC subunit expression pattern in single Purkinje neurons of the wild type
mice ...............................................................................................................