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Laser Capture Microdissection of muscle fiber 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
To locate gene products in distinct muscle fiber populations of different muscle allotypes, a novel protocol was established. A rapid method to distinguish muscle fiber types using histochemistry and immunohistochemistry was used to provide criteria for their selective isolation by laser capture microdissection while preserving messenger RNA (mRNA). Polymerase chain reaction showed a differential expression pattern of muscle specific genes in different muscle fibers in laser captured material.


Introduction
While vertebrate limb muscles have traditionally been the paradigm for studies of skeletal muscle myogenesis, a few atypical muscle groups such as extraocular muscles (EOMs) have distinct functional specializations and patterns of gene expression1. Differences between muscle groups (called allotypes) have been the subject of research for the past decades. The limb and EOM allotypes differ in their pattern of innervation. All limb fibers and 80% of EOM fibers are singly innervated fibers (SIFs) which have only one neuromuscular junction. About 20% of EOM fibers are multiply-innervated fibers (MIFs): they have multiple neuromuscular junctions2. In the past, immunohistochemistry and in situ hybridization were mainly used to study differences between muscle fiber populations3,4, While useful, these techniques are limited in their capability to assess multiple gene products from a mixed muscle fiber population. Moreover, the mixed fiber populations of these muscles make it impossible to study the composition of individual fiber types. To overcome these problems, laser capture microdissection was used to isolate different muscle fiber populations and analyze multiple gene products by polymerase chain reaction. The muscle fiber populations in EOM and limb muscle were distinguished by a combination of the histochemical acetylcholine esterase stain by Karnowsky and Roots5 and immunohistochemical staining with a mouse-monoclonal slow myosin heavy chain (MyHC) antibody (NOQ7-5-4D)6. SIFs show a large, C-shaped neuromuscular junction after acetylcholine esterase staining and do not react with the anti-slow MyHC antibody. The neuromuscular junction of MIFs is smaller and more circular and all MIFs are positive for slow MyHC by immuno-staining. Since mRNA is sensitive to degradation, total time from thawing the slides to dehydration by xylenes was reduced to 32 minutes by combining the histochemical and immunohistochemical reactions and the direct coupling of the antibody to Alexa Fluor 488 by Molecular Probes ZenonTM technology.

This protocol describes a way to isolate and visually distinguish muscle fiber population from two different muscle allotypes and characterize different muscle fibers by multiple RT-PCRs.


Equipment and Reagents
This protocol requires the following reagents:
◊ RNase Away (Invitrogen, Cat. # 10328-011)
◊ ZenonTM Alexa Fluor 488 Mouse IgG1 Labeling Kit (Molecular Probes, Cat. # Z-25002)
◊ PicoPureTM RNA Isolation Kit (Arcturus, Cat. # KIT0204)
◊ RiboAmpTM RNA Amplification Kit (Arcturus, Cat. # KIT0201)
◊ Slow Myosin MyHC Antibody NOQ7-5-4D
◊ PCR Primer (Invitrogen, custom-made)
◊ Goat Serum (Sigma, Cat. # G-9023)
◊ Ethanol (Pharmaco, Cat. # 111ACS200)
◊ Hydranal-Xylenes (Riedel-de Haen, Cat. # 37866)
◊ 2-Methylbutane (Fisher, Cat. # A-03551-4)
◊ Colorfrost /Plus Microscope Slides (Fisher, Cat. # 12-550-19)
◊ Acetylthiocholine Iodide (Sigma, Cat. # A-5751)
◊ Sodium Acetate (Sigma, Cat. # S-2889)
◊ Sodium Citrate (Fluka, Cat. # 71402)
◊ Cupric Sulfate (Sigma, Cat. # C-1297)
◊ CapSure HS LCM Caps (Arcturus, Cat. # LCM0214)
◊ FastStart Taq DNA Poymerase (Roche, Cat. # 2 032 926)
◊ dNTP Mix PCR Grade (Invitrogen, Cat. # 18427-013)
◊ UltraPureTM Agarose (Invitrogen, Cat. # 15510-027)
◊ UltraPureTM 10x TAE (Invitrogen, Cat. # 15558-026)
◊ Ethidium Bromide (Sigma, Cat. # E-1510)
◊ Tissue-Tek OCT Compound (Sakura, Cat. # 4538)
◊ DEPC Treated Water (Ambion, Cat. # 9920)


Equipment and Labware

◊ General equipment necessary:
◊ Disposable gloves
◊ Flow hood
◊ Dry ice
◊ Liquid nitrogen
◊ Ice or cold block
◊ Nuclease-free, aerosol resistant tips
◊ RNase-free microcentrifuge tubes
◊ Scale
◊ KimwipesTM or similar lint-free towels

The following laboratory equipment is required to microdissect muscle fiber properly:
◊ Cryostat
◊ Cryostat knife
◊ Arcturus PixCell II Laser Capture Microdissection System with fluorescence package
◊ QImaingTM Retiga 1300 cooled mono 12-bit digital camera

The following laboratory equipment is 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 System (Bio-Rad, Cat. # 170-8615)


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 manufacturers 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 changed and RNase-free plasticware used.
3. Heat inactivate serum.
4. Use DEPC-treated water for the preparation of all staining solutions and for washing.
5. Use chemicals in the highest grade available.
6. Staining and washing time is to be reduced to a minimum, while still preserving the capability to distinguish different muscle fiber populations.


Method
1. After dissection, limb muscle and EOMs are covered in OCT compound and successively frozen in cooled 2methylbutane (30 seconds) and liquid nitrogen (10 minutes). The muscle can be stored at -80C.

2. The muscle is cut into 10 m sections with the cryostat. During the cutting process, the slides with 4 sections each are stored on dry ice and are afterwards stored at -80C.

3. For the acetylcholine esterase stain by Karnowsky and Roots, the following solution is prepared in the following order:
5 mg of acetylthiocholine iodide are dissolved in 6.5 ml of 0.1 M sodium acetate buffer pH 5.2, followed by:
0.5 ml 0.2 M sodium citrate,
1.0 ml of 30 mM cupric sulfate,
1.0 ml DEPC-treated water, and
1.0 ml of 5 mM potassium ferricyanide.

Larger quantities of the four solutions can be prepared and stored at 4C. But the staining solution has to be prepared fresh since the acetylthiocholine starts to precipitate out of solution after 2 hours.

4. To stain slow MyHC positive fibers rapidly Molecular Probess ZenonTM antibody-labeling technology is used. 20 l of NOQ7-5-4D are mixed with 5 l of Component A (ZenonTM mouse labeling reagent), incubated for 5 minutes. Followed by 5 minutes of incubation with Component B (ZenonTM mouse blocking reagent).

5. After adding Component B to NOQ75-4D, the slide for laser capture microdissection is placed in DEPC-treated water with 2% heat inactivated goat serum for 5 minutes to block non-specific binding sites.

6. The Alexa Fluor 488 conjugated NOQ7-5-4D antibody is diluted 1:40 in the prepared acetylcholine esterase staining solution.

7. The blocked slide is now incubated with the prepared staining solution for 20 minutes.

8. After 20 minutes, the slide is washed twice for 3 minutes in DEPC treated water.

9. The slide is dehydrated in 75%, 90%, and 100% ethanol for 30 seconds respectively, followed by 5 and 7 minutes in fresh xylenes. After 1 minute in a flow hood the slide is ready for laser capture microdissection.

10. The Arcturus PixCell II Laser Capture Microdissection System with fluorescence package is switched on and the blue filter cube is set. The QImaingTM Retiga 1300 cooled mono 12-bit digital camera is activated.

11. The microdissection laser is set to 7 m size, 70 mW power and 850 s pulse duration. The last two values are adjusted to the lowest values possible to still perform laser capture microdissection. This adjustment to a minimal setting enables to actually dissect single muscle fibers.

12. SIFs were dissected based on their large neuromuscular junction stained by Karnowsky and Rootss acetylcholine esterase stain and their lack of slow MyHC staining (Figure 1C). MIFs were dissected based on their smaller neuromuscular junction and a positive signal for slow MyHC (Figure 1A). Additionally, muscle fibers were dissected which were positive or negative for slow MyHC, but didnt show a site of innervation based on Karnowsky and Rootss acetylcholine esterase stain. To dissect single fibers, it can be advantageous to search for fibers on the edge of a fiber bundle as they prove to be easier to dissect without picking up undesired fiber populations.


13. To avoid cross-contamination by other cell types, every cap is scanned visually after the dissection by placing the cap on a fresh slide and viewing it at lowest power. This is followed by a scan at the highest power to make sure that only the desired tissue was captured based on the fluorescence signal (Figures 1B and 1D). The cap is discarded, if unwanted tissue was present. Monochrome pictures of samples were taken using the QImaingTM Retiga 1300 cooled mono 12-bit digital camera.

14. Each cap is then placed in an ExtracSureTM device. The RNA from approximately 25 fibers of each population is pooled and RNA extraction is performed with the PicoPureTM RNA Isolation Kit as described in its protocol within 2 hours of the dehydration with xylenes.

15. After the RNA is extracted, the RiboAmpTM RNA Amplification Kit is used according to the manufacturers instructions to linearly amplify the sample RNA and transcribe the aRNA back to single stranded cDNA. One round of linear amplification was performed for all samples, typically yielding 300 - 600 ng of aRNA.

16. To study expression of muscle specific genes, 50 ng cDNA are amplified with FastStart Taq DNA Polymerase for 40 cycles with conditions and reagents as presented in Table I. The epsilon- and gamma- subunit of the acetylcholine receptor and slow MyHC are amplified. The housekeeping gene GAPDH was amplified as a reference for the amount loaded and the quality of the cDNA. All primers (Table II) are intron spanning to produce a cDNA specific PCR product. To avoid false positive results by PCR product carry-over, PCRs are setup in a flow hood.


17. 15 l of each PCR product were loaded on a 2% agarose gel and separated by 120 V for 25 minutes. Gels are stained with ethidium bromide and visualized using the Gel Doc 2000TM Gel Documentation System.


Results
The data presented in this application note demonstrates that it is possible to isolate a single muscle fiber type or a distinct muscle fiber population and analyze from it the expression of muscle specific genes by RTPCR. Figure 1 demonstrates the isolation of an individual fiber type by LCM. The ZenonTM technology based immunohistochemical staining of slow MyHC positive fibers gives a strong, unambiguous signal in less than 35 minutes. The acetylcholine esterase stain of Karnowsky and Roots allows us to assess rapidly the innervation pattern of muscle fibers. When used alone, this esterase stain can provide results within 5-10 minutes. Hence, rapid identification of distinct fibers based on their immunohistochemistry and innervation pattern allows us time to process tissue via LCM and isolate mRNA without significant time for degradation.

Using this type of analysis on limb muscle confirms 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 sufficient to distinguish muscle fiber types and give new insight into the differences among muscle allotypes.


References
1. Fischer MD, Gorospe JR, Felder E, Bogdanovich S, Pedrosa-Domellof F, Ahima RS, Rubinstein NA, Hoffman EP, Khurana TS. Expression profiling reveals metabolic and structural components of extraocular muscles. Physiol. Genomics. 2002;9:71-84.

2. Bormioli SP, Torresan P, Sartore S, Moschini GB, Schiaffino S. Immunohistochemical identification of slow-tonic fibers in human extrinsic eye muscles. Invest. Ophthalmol. Vis. Sci. 1979;18:303-6.

3. Rubinstein NA, Hoh JF. The distribution of myosin heavy chain isoforms among rat extraocular muscle fiber types. Invest. Ophthalmol. Vis. Sci. 2000;41:3391-8.

4. Brenner HR, Witzemann V, Sakmann 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.

6. Narusawa M, Fitzsimons RB, Izumo S, Nadal-Ginard B, Rubinstein NA, Kelly AM. Slow myosin in developing rat skeletal muscle. J. Cell. Biol. 1987;104:447-59.


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