Mu-opioid receptor expression in neuronal tissues of the rat – a practical course
Type of class Practical course
Offered by Medical Neurosciences
Instructor Dr. rer. nat. Melanie Busch-Dienstfertig
Schedule TWO WEEKS BLOCK IN SS2010, Mon – Fri, 9 a.m. to 5 p.m., to be chosen by students
Location
Klinik für Anaesthesiologie und operative Intensivmedizin
Forschungseinrichtung für Experimentelle Medizin (FEM)
Charité Campus Benjamin Franklin
AG Prof. Stein
Krahmerstr. 6-10
12207 Berlin
Seminar room, labs IV110 and IV112
Target audience MSc Medical Neurosciences; max. 4 students (!)
Learning outcome
Students will analyze neuronal tissues such as cerebellum, hypothalamus, spinal chord, dorsal root ganglia, and peripheral nerve tissue for mu-opioid receptor expression. We will work in a group but each student will be performing all experiments independently. Tissues will be used to isolate total RNA or protein. The students will learn how to synthesize cDNA, to perform conventional RT-PCR, and, as far as possible real-time LightCycler PCR. Protein expression will be analyzed by Western Blotting.
Structure
1st week
- Day 1: Introduction on the mu-opioid receptor (distibution, fuction), on mRNA expression and PCR, and on protein expression and Western Blotting.
- Day 2: Tissue preparation, RNA isolation, and cDNA synthesis
- Day 3: RT-PCR of mu-opioid receptor mRNA and GAPDH mRNA, agarose gel electrophoresis
Excursus: primer design - Day 4: Real-time PCR analysis of mu-opioid receptor mRNA and GAPDH mRNA
- Day 5: Data analysis
2nd week
- Day 1: Protein isolation from tissue, photometric protein quantification (Bradford method), and preparation of polyacrylamide gels
- Day 2: Electrophoresis and blotting
- Day 3: Immunoblotting (anti-mu-opioid receptor)
- Day 4: Immunoblotting (anti-actin)
- Day 5: Data analysis and final discussion
Readings
- Schnell and Wessendorf 2008. J Comp Neurol, Vol. 508, Issue 4, pp 542–64
- Bodnar RJ 2009. Peptides, Vol. 30, Issue 12, pp 2432–79
- Waldhoer et al. 2004. Annu Rev Biochem, Vol. 73, pp 953–90
- Minami and Satoh 1995. Neurosci Res, Vol. 23, Issue 2, pp 121–145
- Elde et al. 1995. Ann N Y Acad Sci, Vol. 757, pp 390–404