Protocols for LCM preparation and analysis
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I. Preparation, LCM and RNA/DNA extraction of Frozen Tissue Sections
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A. Embedding
- 1. Place an empty, labeled cryomold on dry ice for 1 min. It should remain on dry ice during the entire embedding procedure.
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2. Cover the bottom of the cryomold with embedding medium (ie O.C.T.)
- 3. Place the frozen tissue against the bottom. To facilitate cutting, the tissue should be relatively small (1 cm3) and the desired cutting surface should be flush against the bottom.
- 4. Fill the cryomold with embedding medium. Cover the dry ice container and allow the O.C.T. to harden (it will turn white when frozen).
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5. Wrap the block in foil and place at -80 o until cutting.
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B. Cutting
- 1. Remove the block from the cryomold (if not already done) and attach it to the chuck in the cryostat with O.C.T. The cutting surface should be as parallel as possible.
- 2. Allow the block to equilibrate to the cryostat temperature (-20 o) for about 15 minutes. If the block is too cold during cutting this time may need to be extended.
- 3. Cut 10 µm (or thinner) sections onto plain uncoated glass slides. If necessary the sections may be cut thinner or thicker.
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4. Keep the slides in the cryostat or on dry ice if LCM is to be performed that day. Alternatively, they may be stored in paper slide boxes at -80 o until needed.
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C. Staining
Staining should be performed as close as possible to the scheduled LCM transfer time using solution baths that are replaced regularly . Staining should be performed as follows:
- 1. 70% ethanol fix
- 2. purified water wash
- 3. Mayer's hematoxylin (1-2 min.)
- 4. purified water wash
- 5. Blueing reagent (30-60 sec)
- 6. 70% ethanol wash
- 7. 95% ethanol wash
- 8. Eosin Y (1-2 min.)
- 9. 95% ethanol wash (x2)
- 10. 100% ethanol wash
- 11. xylene wash for 1 min
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12. air dry for at least 2 min. to allow the xylene to evaporate completely.
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D. Laser Transfer
For optimal transfer of frozen tissue sections it is best to keep sections < 10 µm thick. Thicker sections are more difficult to visualize. If there are folds in the tissue the cap may not make direct contact with the enitre surface at that area. Therefore it is advisable to inspect the tissue before placing down the cap. If any tissue seems to be mounded or folded, it is best not to place the cap over that area.
The tissue section must be dry and not coverslipped for effective LCM transfer. The staining appears darker and more granular due to light scattered from the irregular air-tissue interface. The tissue where the polymer melts and bonds after laser activation appears lighter and resembles a coverslipped slide due to the replacement of the air in the tissue with the polymer. This phenomenon is called index-matching or "polymer wetting."
Poor transfers may result if the slide is not fully dehyrated (i.e. the 100% ethanol becomes hydrated after repeated use). The final xylene rinse facilitates the efficiency of transfer with LCM. If a tissue section does not transfer well, a longer xylene rinse may help. While other staining protocols can be used, the slides should be dehydrated in a final xylene step.
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E. Sample DNA Extraction Protocol of LCM tissue and PCR as example of DNA analysis
After microdissection (roughly 500-1,000 cells), the cap is inserted into an Eppendorf tube containing digestion buffer (50 ul buffer containing 0.04% Proteinase undefined, 10 mM Tris-HCL (pH 8.0), 1 mM EDTA, and 1% Tween-20). The tube is placed in a 370C oven to equilibriate. It is then placed upside down so that the digestion buffer contacts the tissue on the cap. The incubation continues overnight at 370C. The tube is centrifuged for 5 minutes and the cap is removed. The reaction is heated to 950C for 8 min to inactivate the proteinase K. It can then be used directly as template for PCR (please see PCR section of RT-PCR below). We have used this extraction method for the following methods: LOH analysis, dideoxy fingerprinting (DDF), clonality analysis (chromosome X inactivation) and direct sequencing of PCR products for single base mutational analysis.
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F. Sample RNA Extraction Protocol of LCM tissue and RT-PCR as example of analysis
The cap post laser transfer should be placed tightly onto the Eppendorf tube. The tube is then inverted back and forth over the course of 2 minutes. It is then quick spun to collect all of the buffer. The cap may be removed and replaced with another LCM cap or the investigator may proceed with the RNA extraction. Roughly 1,000-5,000 cells are extracted for most RNA applications. While not quantitative, the intensity of the pink color of the extraction buffer also enables the investigator to gain a sense of the amount of tissue dissected.
It is important to DNAase microdissected samples for applications where DNA would interfere. These include RT-PCR with primers that would amplify DNA and cDNA library construction involving an adapter ligation step. The DNAase used should be certified RNAase free. We have used this protocol to obtain RNA that was subsequently used in the following protocols: RT-PCR, cDNA library construction (with adapter ligation or homopolymer tailing methods), cDNA microarray probe and modified differential display. The RT-PCR protocol described below is for both random primed RT and oligodT (for enrichment of mRNA and 3' message analysis) RT. The (+) RT means that reverse transcriptase is added to the RT reaction. The (-) RT means that the reverse transcriptase is replaced by water. By running a (-) RT it is possible to discern whether the primers used for PCR could be amplifying DNA. There would be no cDNA in the (-) RT reaction. Therefore any amplified product must be due to DNA.
The PCR protocol presented here includes incorporation of radioactivity into the PCR products. For highly abudant cDNAs or clear-cut PCR products it may be possible to discern products on an ethidium bromide treated agarose gel (replace P32 volume in protocol below with water). However, for low abundant transcripts or when PCR product patterns are complicated (i.e. polymorphic markers for LOH), visualization on acrylamide with radioactive incorporation may be necessary. It is listed that 0.2 µL of 20uCi/µl P-32 per 10 µL PCR reaction is used. This amount of radioactivity often results in visible products in less than 2 hrs exposure. It is possible to reduce the radioactivity to 0.05 µL P32 per 10 µL PCR reaction. The exposure times will be increased.
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A. Embedding
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II. Preparation and LCM of Paraffin Embedded Tissue Sections
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A. Fixation
Small pieces of tissue are often embedded in paraffin wax prior to cutting and mounting onto glass slides. The advantages of paraffin embedding over freezing tissue include improved histology and convenient storage. Prior to embedding, however, tissue must be "fixed" to preserve cellular morphology and prevent autolysis. While the routine fixative formalin preserves the tissue morphology, it cross-links RNA and DNA to protein limiting the analysis of nucleic acids. (Mies 1994) DNA derived from microdissected tissues fixed in formalin are still amenable to PCR amplification. The amplification products will be reduced in quantity and size compared to frozen tissues. We routinely amplify 200 bp products from formalin fixed paraffin embedded tissues using the DNA extraction procedure outlined above. Alcohol based fixatives improve the integrity and recovery of the nucleic acids (Foss 1994) and transfer well by LCM.
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B. Processing, Embedding and Tissue Sectioning
Once fixed, the tissue is embedded in paraffin. Paraffin processing can be performed via a routine overnight or accelerated cycle in an automated tissue processer. Once embedded, thin sections are mounted onto glass slides. Consistent LCM transfers have been demonstrated from 5-10 µm thick paraffin embedded tissue sections. For a successful LCM transfer, the strength of the bond between polymer film and the targeted tissue must be stronger than that between the tissue and the underlying glass slide. Therefore, for most tissue types, techniques which increase the adhesion of the tissue section to the glass slide should be avoided. These include the sectioning of tissue onto charged or positive glass slides, and the use of an adhesive in the water bath prior to tissue mounting.
To prevent cross-contamination while sectioning, paraffin and tissue fragments should be wiped from the area with xylenes between each slide. If possible, a fresh microtome blade should be used for each block. The prepared tissue slides should be stored at room temperature until required for LCM transfer.
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C. Staining
Staining should be performed as close as possible to the scheduled
LCM transfer time using solution baths that are replaced regularly .
- 1. xylenes to deparaffinize the slides (5 min x 2)
- 2. 100% ethanol wash
- 3. 95% ethanol wash
- 4. 70% ethanol wash
- 5. purified water wash
- 6. Mayer's hematoxylin (1-2 min.)
- 7. purified ater wash
- 8. Bluing reagent (30-60 sec)
- 9. 70% ethanol wash
- 10. 95% ethanol wash
- 11. Eosin Y (1-2 min.)
- 12. 95% ethanol wash (x2)
- 13. 100% ethanol wash
- 14. xylene wash for 1 min
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15. Air dry for at least 2 min. to allow the xylene to evaporate completely.
RNA Isolation (modified Stratagene Microisolation protocol)
- Place the cap in an eppendorf tube containing 200 µL RNA denaturing buffer (GITC) and 1.6 µL b-mercaptoethanol. Invert several times over the course of 2 minutes to digest the tissue off of cap.
- Remove the solution from the ependorf tube and replace it in a sturdy 1.5 mL tube.
- Add 20 µL (0.1X volume) 2M sodium acetate (PH 4.0)
- Add 220 µL (1X volume) water saturated phenol (bottom layer)
- Add 60 µL
- Add 60 µL(0.3X volume) chloroform-isoamyl alcohol
- Vortex vigorously
- Put on wet ice for 15 min
- Centrifuge for 30 min at 4C to separate the aqueous and organic phases.
- Transfer upper aqueous layer to a new tube
- Add 1 µL glycogen (10 ug/ul). Glycogen is a carrier that is used if RNA quantities are less than 1 ug. It also facilitates visualization of the pellet.
- Add 200 µL cold isopropanol
- Put in -800C freezer for at least 30 minutes. It may be left overnight.
- Centrifuge for 30 min at 40C with caps hinges pointing outward so that the location of the pellet can be better predicted
- Remove the majority of the supernatant with a 1000 µL tip and then switch to a smaller pipet to remove the rest of the supernatant. This minimizes disruption of the RNA pellet.
- Wash with 70% Ethanol. Add the alcohol and spin for 5 min at 40C.
- Remove the supernatant as explained above. All of the supernatant should be removed at this point.
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Let the pellet air dry on ice to remove any residual ethanol.
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Can store pellet at -800C until use.
- To RNA pellet add 15 µL DEPC water, 1 µL 20 U/µL RNAase inhibitor (Perkin Elmer), 2 µL 10X DNAase buffer (Genhunter) and 2 µL 10 U/µL DNAase (20 units total).
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Incubate at 370C for 2 hrs
Re-extracton of RNA (same reagents as above)
- 2 uL NaOAc
- 22 uL Phenol
- 6 uL Chloroform-isoamyl alcohol
- Place on ice for 15 min
- Centrifuge 10 min at 40C
- Transfer upper layer to a new tube
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Continue with RNA extraction from step 10
Reverse Transcription (final volume 20 uL)
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Add 24 uL water and 1uL 20 U/µL RNAase inhibitor to RNA pellet. Resuspend RNA pellet well.
- Aliquot 12 uL into 2 tubes for the (+) and (-) RT's.
- Add 4 µL 5X RT buffer (Genhunter or other manufacturer)
- Add 2 µL dNTP (250 µM) (Genhunter)
- Add 1 uL 10 µM random hexamer primers (Perkin Elmer -PE) or 2 µL 10µM oligodT primers
- Incubate 5 min at 65 0C
- Incubate 10 min at 37 0C for oligodT (10 min @ 250C if using random hexamer primers)
- Add 1 µL 100 units/uL MMLV reverse transcriptase to the (+) RT tubes ONLY . Add 1 µL water to the (-) RT tubes. (If random priming incubate another 10 min @ 250C).
- Heat 50 min at 370C (40 min at 370C if random priming)
- 5 min at 950C
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The cDNA may be stored at -200C until use
Components and cycling will depend on individual template and primers
- 1.0 µL cDNA (or DNA)
- 1.0 µL 10X PCR Buffer
- 0.8 µL dNTP (25 µM)
- 0.2 µL primer 1 (10 µM)
- 0.2 µL primer 2 (10 µM)
- 0.2 µL P-33 or P-3undefined(20 µCi/ul)
- 0.2 µL 5 U/ul Taq DNA polymerase
- 6.4 µL DEPC-H20
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10.0 µL
Sample Temps/Times for PCR in PE 9600 thermocycler
- - 940C 2 min
- - 35 cycles of: 940C 45 sec (or shorter)
- X 0C 45 sec (X is annealing temperature)
- 720C 2 min
- - 720C 10 min
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- 40C forever
P.A.G.E. *(onlyif radioactivity is used)
- While the PCR is cycling, pour a 6% Polyacrylamidesequencing gel (or use Novex minigel)
- After cycling is completed add 10µL 2X loading dye to samples
- Denature samples at 95 C for 3 minutes and place directly on ice
- Load 3.5 µL sample on gel
- Run at 1600 volts to desired distance as trackedby dye lines
- Dry gel (or mount Novex)
- Expose to film in cassette with intensifying screen
- Develop film
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A. Fixation
