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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PURIFICATION: EXTRACT FROM CELLS
PROCESSING OF MICRODISSECTED TISSUE FOR MOLECULAR ANALYSIS: DNA-BASED STUDIES, RNA-BASED STUDIES, AND PROTEIN-BASED STUDIES
Processing of Microdissected Tissue for Molecular Analysis: DNA-based Studies, RNA-based Studies, and Protein-based Studies
Contributor: The Molecular Profiling Initiative of the National Cancer Institute
URL: Molecular Profiling Initiative
This protocol describes molecular analysis of cells isolated by microdissection. Microdissection isolates cells from tissue samples that have been prepared for visualization with a light microscope. After microdissection of tissue (see Protocol ID#2235, Protocol ID#2236, Protocol ID#2237), cells of interest are collected and processed for molecular analysis. This protocol describes the procedures for the isolation of DNA, RNA, or protein from selected cells (see Hint #1). Procedure
Isolation of DNA from Microdissected Cells
A. More than 10,000 Cells
1. If the amount of microdissected material is substantial (>10,000 cells), then any of the standard procedures for isolating DNA are acceptable (see Protocol ID#90, Protocol ID#178 and Protocol ID#1221).
B. Less than 10,000 Cells
1. If the number of cells procured is minimal (less than 10,000 cells), then a simple proteinase K treatment prior to analysis by PCR is recommended. Procured cells are suspended in Buffer #1 at a concentration of approximately 20 cells per μl.
2. Incubate the suspended cells at 37°C overnight.
3. Boil the sample for 8 min to inactivate the Proteinase K. The sample is now ready for analysis by PCR (see Hint #2).
C. Formalin-fixed, Paraffin-embedded Tissue
1. DNA can be amplified by PCR with DNA recovered from standard formalin-fixed, paraffin-embedded tissue. This allows investigators to perform studies on archived tissue, such as biopsy samples from patients. These studies are often technically challenging. The type of fixative used and the fixation time impact heavily on the quality of the DNA recovered after microdissection, resulting in amplifications that are widely disparate in yield among samples in a study (see Hint #3).
D. Other Fixatives
1. Fixatives containing heavy metals or low pH should be avoided. Application of non-formalin based fixatives generally results in recovery of higher quality DNA.
1. Careful but rapid tissue processing is important in the preservation of molecular materials. Tissue should be processed immediately after their isolation (e.g., biopsy tissue removal from a patient). Thin sections should be prepared to enable rapid penetration of the fixative. Fixation for the optimal amount of time is critical, and over-fixation (>24 hours) should be avoided.
F.Total RNA Isolation (see Hint #4)
1. Before beginning, prepare all equipment and solutions for RNA work (see the BioTools section of the website).
2. Place the laser capture microscopy (LCM) cap (see Protocol ID#2237) containing isolated cells in a microcentrifuge tube containing 200 μl of RNA Denaturing Buffer (Statagene) and 1.6 μl of 2-Mercaptoethanol. Invert the tube several times for 2 minutes to digest the tissue off the cap (see Hint #5).
3. Transfer the solution from the tube into a fresh RNase-free 1.5 ml microcentrifuge tube.
4. Add 20 μl of 2 M Sodium Acetate, pH 4.0.
5. Add 220 μl of Water-saturated Phenol.
6. Add 60 μl of Chloroform/Isoamyl Alcohol.
7. Mix vigorously by vortexing for 15 sec.
8. Incubate on ice for 15 min.
9. Centrifuge in a microcentrifuge at maximum speed for 30 min at 4°C to separate the aqueous and organic phases.
10. Transfer upper aqueous layer to a fresh tube (see Hint #6).
11. Add 2 μl of Glycogen and an equal volume (200 to 300 μl) of ice-cold Isopropanol to the aqueous layer (see Hint #7).
12. Invert the tube several times to mix. Incubate the mixture at -80°C for at least 30 min to overnight.
13. Centrifuge for 30 min at 4°C with the cap hinges pointed outward so that the location of the pellet can be identified.
14. Discard the supernatant and wash the pellet with 300 μl of ice-cold 70% (v/v) Ethanol. Add the Alcohol and centrifuge at maximum speed for 5 min at 4°C.
15. Discard the supernatant and let the pellet air-dry for 10 min to evaporate any residual Ethanol. Do not allow the pellets to dry for longer periods of time.
16. The pellet may be stored at -80°C until needed, or proceed to DNase step.
Optional DNase Treatment (see Hint #8)
17. Add 15 μl of DEPC-treated ddH2O and 1 μl of RNase Inhibitor (Perkin Elmer) to the RNA pellet.
18. Gently mix the resuspended RNA by flicking the tube until the pellet is dissolved.
19. Centrifuge the tube for 10 sec at maximum speed to force all of the liquid to the bottom of the tube.
20. Add 2 μl of 10X DNase Buffer (GenHunter) and 2 μl of DNase (GenHunter).
21. Incubate the tube at 37°C for 2 hr.
22. Re-extract the RNA by adding:
2 μl of 2 M Sodium Acetate, pH 4.0
22 μl of water-saturated Phenol
6 μl of Chloroform/Isoamyl Alcohol.
23. Mix vigorously by vortexing for 15 sec.
24. Incubate on wet ice for 15 min.
25. Centrifuge at maximum speed in a microcentrifuge for 10 min at 4°C.
26. Transfer the upper aqueous layer to a fresh tube (see Hint #6).
27. Continue with RNA extraction from Step #F11 in Total RNA Isolation.
1. The methodology and buffers utilized for processing microdissected samples for protein-based studies are variable and depend on the downstream molecular analysis method. Caps can be directly resuspended and boiled in SDS-PAGE loading buffer to lyse cells for SDS-PAGE (see Protocol ID#2247). According to the example from the contributor's lab, the recovered cells are placed in the following buffer for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) studies:
100 μl of IEF Lysing Solution containing 7 M Urea
2 M Thiourea
4% (w/v) CHAPS
1% (w/v) MEGA-10
1% (w/v) Octyl-β-Glucopyranoside
40 mM Tris
50 mM DTT
2 mM Tri-butyl Phosphine (TBP)
0.5% (v/v) Pharmalytes (pH ranges 3-10, 4-7, or 6-11).
2. The buffer is applied directly to the microdissected cells adhered on the LCM cap, placed into a microcentrifuge tube, and mixed vigorously by vortexing for one min to lyse the cells.
H. Limitations of Two Dimensional (2D) SDS-PAGE Analysis of Microdissected Cells
1. Sensitivity: Currently, it is possible to analyze on the order of 50,000 cells. Approximately 675 distinct human proteins can be visualized from this number of cells by two-dimensional SDS-PAGE after staining with silver. Assuming the lower limit of detection to be one nanogram, the analysis identifies proteins in the abundance range of 50,000 to 1,000,000 copies per cell. This level of sensitivity is sufficient for misregulated expression of high or moderate abundance proteins.
2. Methods to Improve Sensitivity: As a solution, the NCI Prostate Group (see Contributor's website above) has developed methods, such as 125Iodine labeling or protein biotinylation, which dramatically increases the number of proteins detected. Similarly, scanning immunodetection methods with class-specific antibodies allow for sensitive detection of specific subsets of proteins (e.g., all known proteins involved with cell cycle regulation).
3. Protein Identification: Unfortunately, protein fingerprinting is technically difficult due to the small amount of material analyzed and subsequent difficulty in identifying low abundance proteins of interest.
4. Methods to Improve Protein Identification: A strategy to improve protein identification is to compare parallel "diagnostic" and "sequencing" 2D SDS-PAGE profiles from normal and affected tissue samples.
Diagnostic fingerprints are derived from microdissected cells and provide maximal sensitivity for the detection of normal versus tumor differences.
Sequencing fingerprints allows for the determination of protein identity. The sequencing 2D SDS-PAGE profiles are generated from serial, whole tissue section cryostat samples that contain abundant amounts of protein representing all cell types present in the tissue of interest including the dissected cell population(s).
Comparison of the diagnostic and sequencing 2D SDS-PAGE profiles enables the identification of proteins of interest for subsequent sequence analysis.
DNase 10 Units/μl DNase (GenHunter) 10 X DNase buffer (GenHunter) 70% (v/v) Ethanol Glycogen 10 mg/ml Glycogen
Prepare in ddH2O
2 M Sodium Acetate Chloroform/Isoamyl Alcohol 24:1 Chloroform: Isoamyl Alcohol
saturated with ddH2O
Water-saturated Phenol Store at 4°C under dark conditions
Use ddH2O instead of TE to equilibrate
See Protocol ID#578 for preparation
RNA Denaturing Buffer Provided in the Stratagene Microisolation Kit (Stratagene) Buffer #1 10 mM Tris-HCl, pH 8.0
0.1 mg/ml Proteinase K
1 mM EDTA
1% (w/v) Tween 20
0.5% (v/v) Pharmalytes pH ranges 3-10, 4-7, or 6-11 2 mM Tri-butyl Phosphine (TBP) 50 mM DTT store at -20°C 40 mM Tris 1% (w/v) Octyl-β-Glucopyranoside 1% (w/v) MEGA-10 4% (w/v) CHAPS 2 M Thiourea 100 μl of IEF Lysing Solution prepared with 7 M Urea RNase Inhibitor 20 Units/μl RNase Inhibitor (Perkin Elmer) BioReagents and Chemicals
Water saturated Phenol
Tri-butyl phosphine (TBP)
Non-Formalin based Fixatives
Ethylenediamine Tetraacetic Acid
Denaturing Buffer, RNA
1. This method was used successfully with prostate tissue. Investigators must be aware that they will need to tailor the protocol for their own research objectives and particular tissue under study.
2. Longer incubation times, higher incubation temperatures, and higher concentrations of Proteinase K have been reported to improve the quality of DNA recovered from fixed tissue sections.
3. For a target cell type containing very small numbers of cells, the contributor of this protocol recommends the isolation of as many consecutive histological samples as possible to maximize the total number of cells procured. There is no optimal number of cells that should be collected from a microdissection since results vary significantly depending on the tissue source. For frozen tissue, approximately 20 cells/μl of Buffer #1 is recommended as a good starting point; however, more cells per PCR reaction may be needed for DNA recovered from formalin-fixed paraffin-embedded tissue (see Protocol ID#2232).
4. If amplification from a sample does not initially yield a PCR product, a ten-fold dilution of the boiled sample may dilute inhibitors of DNA polymerase from the tissue. Design of PCR primer sets to produce products in the 150 to 200 bp range is beneficial since the DNA is often crosslinked and/or fragmented and may not reliably amplify larger products.
5. Recovery and analysis of RNA from frozen tissue sections can be achieved using slight modifications of standard methods (see Protocol ID#527, Protocol ID#1002). The contributor of this protocol has successfully used the Statagene™ Microisolation Kit to recover total RNA from microdissected samples.
6. Do not transfer any of the organic phase as small amounts will inhibit subsequent enzymatic manipulations. If in doubt, add one volume of 100% (v/v) Chloroform, mix well, and centrifuge for 10 minutes at 4°C to separate the aqueous and organic phases. Transfer the upper layer to a new tube.
7. Glycogen facilitates visualization of the pellet, which can be problematic with small amounts of RNA.
8. DNase treatment is highly recommended for microdissected cells. Genomic DNA contamination is often problematic with these samples, possibly due to the small DNA fragments that are created during tissue processing.