Appnote2.indd

A P P L I C A T I O N N O T E #2
Laser Capture Microdissection of muscle fi ber populations
and expression analysis by RT-PCR
Sven Fraterman and Neal Rubinstein, Department of Cell and Developmental Biology,
University of Pennsylvania School of Medicine, Philadelphia
Abstract
acetylcholine esterase stain by Karnowsky and To locate gene products in distinct muscle with a mouse-monoclonal slow myosin heavy allotypes, a novel protocol was established. SIFs show a large, C-shaped neuromuscular junction after acetylcholine esterase staining and do not react with the anti-slow MyHC immunohistochemistry was used to provide criteria for their selective isolation by laser of MIFs is smaller and more circular and capture microdissection while preserving immuno-staining. Since mRNA is sensitive reaction showed a differential expression pattern of muscle specifi c genes in different the slides to dehydration by xylenes was muscle fi bers in laser captured material.
Introduction
traditionally been the paradigm for studies Molecular Probes ZenonTM technology.
atypical muscle groups such as extraocular This protocol describes a way to isolate groups (called “allotypes”) have been the subject of research for the past decades.
The limb and EOM allotypes differ in Equipment and Reagents
Equipment and Labware
fi bers and 80% of EOM fi bers are singly innervated fi bers (SIFs) which have only one neuromuscular junction. About 20% of EOM fi bers are multiply-innervated fi bers (MIFs): they have multiple neuromuscular junctions2. In the past, immunohistochemistry used to study differences between muscle fiber populations3,4, While useful, these techniques are limited in their capability mixed muscle fi ber population. Moreover, To overcome these problems, laser capture microdissection was used to isolate different multiple gene products by polymerase chain reaction. The muscle fi ber populations in 6 5 0 . 9 6 2 . 3 0 2 0 t e l 8 8 8 . 4 6 6 . 7 9 1 1 t o l l - f r e e 6 5 0 . 9 6 2 . 3 0 3 9 f a x t e c h s u p p o r t @ a r c t u r . c o m w w w . a r c t u r . c o m A P P L I C A T I O N N O T E #2
required for RNA isolation and analysis by RT-PCR: Incubation oven Microcentrifuge Arcturus alignment tray Thermo Cycler with heated lid Horizontal gel chamber Power supply Gel Doc 2000TM Documentation RNase-free Technique
In addition to the usual precautions listed below, some special precautions were taken connected to the rapid staining protocol: 1. Use RNase® AWAY according to the manufacturer’s instructions on laboratory bench surfaces, cryostat, cryostat knife, PixCell® II Laser Capture Microdissection System and Arcturus alignment tray.
2. Disposable gloves are to be frequently Figure 1. Laser Capture Microdissection of Muscle Fiber Populations.
A multiply innervated muscle fi ber is shown before (A) and after (B) laser capture mircodissection. The fi ber shows
strong immunohistochemical staining for slow myosin and the two grappe-like neuromuscular junctions are Figures C and D show the laser capture microdissection process of a population of singly innervated muscle fi bers. This preparation of all staining solutions and fi ber population is negative for slow MyHC immuno-staining and show comparable large neuromuscular junctions which are again marked with an arrow. Figures A and C are taken with the fl uorescence and dimmed visual light on and Figures B and D show the fi ber after microdissection on the laser microdissection cap with only fl uorescence light on. Some autofl uoresence is detectable in Figure D, but the signal is weaker than in Figure B.
preserving the capability to distinguish different muscle fi ber populations.
• 5 mg of acetylthiocholine iodide are 2. The muscle is cut into 10 µm sections the staining solution has to be prepared fresh since the acetylthiocholine starts process, the slides with 4 sections each are stored on dry ice and are afterwards 3. For the acetylcholine esterase stain by 6 5 0 . 9 6 2 . 3 0 2 0 t e l 8 8 8 . 4 6 6 . 7 9 1 1 t o l l - f r e e 6 5 0 . 9 6 2 . 3 0 3 9 f a x t e c h s u p p o r t @ a r c t u r . c o m w w w . a r c t u r . c o m A P P L I C A T I O N N O T E #2
with the prepared staining solution for 20 minutes.
8. After 20 minutes, the slide is washed twice for 3 minutes in DEPC treated water.
Figure 2. Semi-quantitative RT-PCR of different muscle fi ber populations.
Gel pictures of polymerase chain reaction for epsilon- and gamma- subunit of the acetylcholine receptor, slow minute in a fl ow hood the slide is ready Muscle fi bers were distinguished by their reactivity to the acetylcholine esterase stain by Karnowsky and Roots and to Alexa Fluor 488 conjugated slow MyHC antibody. In the leg, three different populations were analyzed: (NMJ), the neuromuscular junction region of fi bers negative for slow MyHC; (slow +), non innervated region of fi bers positive for slow MyHC; (slow -), non innervated region of fi bers negative for slow MyHC. In the EOMs, fi bers were again distinguished by their reactivity to anti-slow MyHC antibody and their staining for acetylcholine esterase. (NMJ-SIF), the neuromuscular junction region of fi bers negative for slow MyHC - these are all singly innervated; (NMJ-MIF), the neuromuscular junction region of fi bers positive for slow MyHC - these are all multiply-innervated; (slow +), non innervated region of fi bers positive for slow MyHC - i.e. MIFs; (slow -), non innervated region of fi bers 11. The microdissection laser is set to 7 the desired tissue was captured based on the fl uorescence signal (Figures 1B and 12. SIFs were dissected based on their large 17. 15 µl of each PCR product were loaded described in its protocol within 2 hours The data presented in this application note demonstrates that it is possible to isolate a single muscle fi ber type or a distinct muscle fi ber population and analyze from it the expression of muscle specifi c genes by RT- esterase stain. To dissect single fi bers, it PCR. Figure 1 demonstrates the isolation of an can be advantageous to search for fi bers individual fi ber type by LCM. The ZenonTM staining of slow MyHC positive fi bers gives picking up undesired fi ber populations.
a strong, unambiguous signal in less than 16. To study expression of muscle specifi c 35 minutes. The acetylcholine esterase stain 13. To avoid cross-contamination by other of Karnowsky and Roots allows us to assess cell types, every cap is scanned visually rapidly the innervation pattern of muscle fi bers. When used alone, this esterase stain on a fresh slide and viewing it at lowest can provide results within 5-10 minutes. power. This is followed by a scan at the Hence, rapid identifi cation of distinct fi bers 6 5 0 . 9 6 2 . 3 0 2 0 t e l 8 8 8 . 4 6 6 . 7 9 1 1 t o l l - f r e e 6 5 0 . 9 6 2 . 3 0 3 9 f a x t e c h s u p p o r t @ a r c t u r . c o m w w w . a r c t u r . c o m A P P L I C A T I O N N O T E #2
Table I. PCR Cycling Conditions, Reaction Components and Volumes Used and innervation pattern allows us time to process tissue via LCM and isolate mRNA FastStart PCR Master Mix
Thermal Cycler programming
without signifi cant time for degradation. Using this type of analysis on limb muscle confi rms data previously obtained by other means4. Hence, we can apply the technique to another muscle allotype, the EOM, and be sure that this protocol and the Arcturus PixCell II Laser Capture Microdissection System is suffi cient to distinguish muscle fiber types and give new insight into Table II. PCR Primer Sequences and Annealing Temperatures Forward primer (fwd)
Annealing
References
size (bp)
Reverse primer (rev)
Physiol. Genomics. 2002;9:71-84.
Immunohistochemical identifi cation of slow-tonic fi bers in human extrinsic eye muscles. Invest. Ophthalmol. Vis. Sci. 1979;18:303-6.
distribution of myosin heavy chain isoforms among rat extraocular muscle fi ber types. Invest. Ophthalmol. Vis. Sci. 2000;41:3391-8.
B. Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature. 1990;344:544-7.
5. Karnowsky MJ, Roots, L.A. A “direct coloring” thiocholine method for cholinesterase. J. Histochem. Cythochem. 1964:219-221.
S, Nadal-Ginard B, Rubinstein NA, Kelly AM. Slow myosin in developing rat skeletal muscle. J. Cell. Biol. 1987;104:447-59.
6 5 0 . 9 6 2 . 3 0 2 0 t e l 8 8 8 . 4 6 6 . 7 9 1 1 t o l l - f r e e 6 5 0 . 9 6 2 . 3 0 3 9 f a x t e c h s u p p o r t @ a r c t u r . c o m w w w . a r

Source: http://alphametrix.at/downloads/LCM_muscle_fiber.pdf