Chapter 8. Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections. Renate Burgemeister. Abstract. 1. Introduction

Transcription

1 Chapter 8 Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections Renate Burgemeister Abstract Tissue heterogeneity is a common source of unsuccessful experiments. Laser capture microdissection is a tool to prepare homogeneous tissue and cell areas as starting material for reliable and reproducible results as it allows the defined investigation of spatially different tissue areas. Nearly all samples allow the extraction of DNA. Fresh or fresh frozen samples are an ideal source for getting access to high-quality RNA. But also the large archives of formalin-fixed, paraffin-embedded (FFPE) tissue specimens are a valuable source of sample material for RNA extraction. Optimized protocols may help to make the RNA from FFPE material suitable for expression studies. Key words: Laser microdissection, Defined tissue areas, Homogeneous tissue, Biomarker identification, High-quality RNA, FFPE tissue, Expression studies, Archived biopsies 1. Introduction Genomics and proteomics techniques have become increasingly sophisticated; however, accuracy and reliability of results are strongly dependent from the purity of the sample. Recent scientific and medical applications depend on the selective procurement of defined cell or tissue populations (1). In the characterization of the early molecular genetic events of tumor development, it is of immense importance to getting access to histopathologically accurately defined tissue. To obtain high-quality DNA, mrna, and proteins from these (often small) tissue samples and even from single cells, laser microdissection is one of the most useful techniques. Microdissected tissue material or single cells, free of contaminating and unwanted tissue components, are extremely important Fahd Al-Mulla (ed.), Formalin-Fixed Paraffin-Embedded Tissues: Methods and Protocols, Methods in Molecular Biology, vol. 724, DOI / _8, Springer Science+Business Media, LLC

2 118 Burgemeister for producing clean data, e.g., biomarker identification in cancer diagnostics and in elucidating biological variance within tumors (2 4). Thus, these technologies will have an enormous impact on molecular pathology with the potential to improve diagnosis, estimation of prognosis, and treatment decisions in individual patients (5). In most instances, RNA can be extracted from fresh frozen material in high quality. Unfortunately sometimes the morphology of this material is inadequate. While formalin-fixation and paraffin-embedding efficiently preserve tissues for morphological analysis, the effects of the fixation process on nucleic acids make molecular analyses difficult. Nucleic acids in FFPE tissues are cross-linked and often irreversibly damaged, becoming increasingly fragmented with prolonged storage. Nevertheless, FFPE tissue samples represent a large source of unused biological material for basic clinical research. In every hospital, there are large archives of morphologically defined biopsies that exist as fixed and embedded samples. Retrospective analysis of this archived material derived from normal and pathologically altered tissues (6, 7), for which clinical data are available, could enable the correlation of molecular findings with the effect of treatment and the clinical outcome (8). Expression studies in these biopsies offer a promising extension of current methods to study the pathogenesis of many different diseases. Laser microdissection allows for the selective isolation of specific tissue or cell areas and is the best option to quantitate mrna levels from such kind of archival material (6 8). 2. Materials 2.1. Removing Coverslips of Archived Samples 2.2. MembraneSlides Xylene or warm water (30 50 C). 1. MembraneSlide 1.0 PEN (Carl Zeiss). 2. MembraneSlide 0.17 PEN (Carl Zeiss). 3. MembraneSlide 1.0 PET (Carl Zeiss). 4. MembraneSlide 0.17 PET (Carl Zeiss) Heat Inactivation to Remove Nucleases Heating oven, 180 C UV Treatment of the Membrane UV light source, 254 nm.

3 Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections 2.5. Poly-l-Lysine Treatment 119 Poly-l-lysine 0.1% (w/v) Mounting FFPE Sections onto MembraneSlides 1. Drying oven, 56 C Staining 1. 70% (v/v) Ethanol. 2. Ethanol 100, 96, 70%. 2. RNase-free distilled water. 3. Cresyl violet acetate solution: Dissolve solid Cresyl violet acetate at a concentration of 1% (w/v) in 50% (v/v) ethanol at room temperature with agitation/stirring for several hours or overnight. Filter the staining solution before use to remove unsolubilized powder (see Note 1). For an enhancement of the staining (see Note 2). 4. Mayer s hematoxylin solution (ready to use). 5. DEPC-treated tap water: 50% (v/v) DEPC in Aqua dest (see Note 3). 6. Eosin Y solution aqueous (ready to use). 7. Increasing ethanol series: 70% (v/v) ethanol, 96% (v/v) ethanol, 100% (v/v) ethanol Dry Collection of Laser Microdissected Material (Recommended for Subsequent RNA Extraction) 1. AdhesiveCap 200 (Carl Zeiss), AdhesiveCap 500 (Carl Zeiss) (see Note 3) Wet Collection of Laser Microdissected Material Capturing buffer: 20 ml of 0.05 M EDTA, ph 8.0, 200 ml of 1 M Tris HCl, ph 8.0, 50 ml Igepal CA-630, and 100 ml of 20 mg/ml Proteinase K (see Notes 5 7) RNA Extraction 2. Removal of RNases from regular tubes with 0.1% (v/v) DEPC. Add 100 ml DEPC to 100 ml of double-distilled water (see Note 4). Stir for 5 6 h at room temperature to dissolve the DEPC. Dump the reaction tubes in the DEPC solution, take care that the tubes are completely covered with liquid (not blistered!) and incubate overnight at room temperature. Autoclave the tubes together with the solution for 20 min at 121 C to inactivate the DEPC. Discard the liquid carefully and thoroughly. Dry the tubes at C. Use the tubes as usual. 1. Incubator, 55 C. 2. Qiagen RNeasy FFPE Kit. 3. Digestion buffer containing Proteinase K: 150 mm NaCl, 100 mm Tris HCl, ph 7.5, 0.5% Igepal, 0.5 mg/ml Proteinase K.

4 120 Burgemeister DNA Extraction 1. Qiagen QIAamp Micro Kit. 2. Digestion buffer containing Proteinase K: 150 mm NaCl, 100 mm Tris HCl, ph 7.5, 0.5% Igepal, 0.5 mg/ml Proteinase K. 3. Methods 1. To allow laser capture microdissection (LCM) (cutting and lifting) a coverslip and mounting medium must not be applied. 2. To ensure RNase-free conditions use only RNase-free solutions and materials. 3. For FFPE samples, a Proteinase K digestion step is essential. The time necessary for optimal Proteinase K digestion depends on many factors such as tissue type, fixation procedure, or element size of lifted material. An overnight digestion (12 18 h) is a good starting point for optimization, but shorter digestion times may be tested as well. To our experience, digestion of at least 3 h should be applied with any extraction procedure. 4. A forecast of the extractable amount of RNA from FFPE tissue is very difficult since many factors such as species, cell/ tissue fixation, staining, fragmentation, modification, and others will strongly influence the outcome. Any FFPE tissue block should therefore be tested individually Removing the Coverslip of Archived Samples 1. To allow LCM (cutting and lifting), a coverslip and mounting medium must not be applied. 2. Depending on the applied mounting medium of archived samples (whether it was xylene based or water soluble), the whole slide should be completely submerged in the respective solvent. 3. Stand up slide in a glass jar filled with either pure xylene or warm water (30 50 C). 4. Let the coverslip swim off (see Note 8). 5. Air-dry the slide MembraneSlides 1. MembraneSlides for LCM are used as regular glass slides. They are glass slides covered with a membrane on one side (Fig. 1). This membrane is easily cut together with the tissue and acts as a stabilizing backbone during lifting. Therefore, even large areas are captured by a single laser pulse without affecting the morphological integrity.

5 Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections 121 Fig. 1. MembraneSlides are glass slides covered with a thin biochemically inert membrane. This warrants excellent DNA, RNA, or protein recovery. 2. Use of MembraneSlide is especially important for isolating single cells, chromosomes as well as live cells, or small organisms. There are slides available in different thickness (1 and 0.17 mm) covered with different membranes [polyethylene naphthalate (PEN)- or polyethylene teraphthalate (PET) membrane] (Carl Zeiss). These membranes are highly absorptive in the UV-A range, which facilitates laser cutting. 3. When working with low magnifying objectives such as 5 or 10, both regular 1- and 0.17-mm thick slides can be used. To keep this flexibility for higher magnifications (20, 40, or 63 ), we recommend using long-distance objectives. With those, it is possible to adapt the working distance to the different slides by moving the correction collar on the objective. Due to the short working distance of the 100 magnifying objectives, only 0.17-mm thin slides can be used for that lens. 4. Besides MembraneSlides, regular glass slides are applicable for laser microdissection. Freshly prepared slides must not be coverslipped. Archived old slides have to be de-coverslipped (see Subheading 3.1). 5. With the laser microdissection system PALM MicroBeam almost every kind of biological material can be microdissected and lifted directly from slides into collection devices. To facilitate easy lifting, additional adhesive substances or Superfrost + charged slides should only be applied when absolutely necessary for the attachment of poorly adhering special material (e.g., some brain sections or blood vessel rings). In those cases, higher laser energy is needed for lifting.

6 122 Burgemeister 3.3. Heat Inactivation to Remove Nucleases 1. To make MembraneSlides or glass slides RNase free, heat slides at 180 C for 4 h. Nucleases are inactivated this way. 2. MembraneSlide NF (nuclease free) is certified to be free of DNase, RNase, and human DNA. Treatments to remove nucleases are therefore not necessary UV Treatment of the Membrane 1. To overcome the hydrophobic nature of the membrane, it for some samples may be advisable to irradiate the MembraneSlide with UV light at 254 nm for 30 min (e.g., in a cell culture hood). The membrane gets more hydrophilic, therefore the adhesion of the sections (paraffin- and cryosections) is improved. 2. Positive side effects are sterilization and destruction of potentially contaminating nucleic acids Poly-l-Lysine Treatment 1. Additional coating of the slide with poly-l-lysine could be beneficial for poorly adhering materials (e.g., brain sections) and should be performed after UV treatment. Distribute a drop of the solution on top of the slide. 2. Let air-dry at room temperature for 2 3 min. Avoid any leakage of the membrane, as this might result in the impairment of LCM Mounting FFPE Sections onto MembraneSlides 1. Sections are mounted onto MembraneSlides the same way as routinely done using glass slides. Floating the section on warm water as well as hot plate techniques can be applied. After mounting, let dry the slides overnight in a drying oven at 56 C. To allow laser cutting and lifting, a coverslip and standard mounting medium must not be applied. 2. Paraffin will reduce the efficiency of the laser, sometimes completely inhibiting cutting and lifting. For using unstained sections, it is therefore very important to remove the paraffin before laser cutting and lifting. If applying standard staining procedures deparaffinization is routinely included in any protocol. 3. Deparaffinization of unstained sections: xylene 2 min, two times, ethanol 100% 1 min, ethanol 96% 1 min, and ethanol 70% 1 min Staining 1. After deparaffinization continue with the staining procedure of your choice. Most standard staining procedures can be used for FFPE sections. Most standard histological stains (e.g., hematoxylin/eosin (HE), Methyl Green, Cresyl Violet, and Nuclear Fast Red) are compatible with subsequent RNA isolation. For best RNA results from stained samples Cresyl Violet or HE staining is recommended. This short staining procedure colors the nuclei violet and the cytoplasm weak violet.

7 Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections 123 It is particularly recommended for RNase-rich tissues since all solutions contain high ethanol. 2. Staining for subsequent RNA extraction: use only freshly prepared and precooled staining solutions. Use RNase-free water and solutions for all steps. All required reagents should be kept on ice. 3. Cresyl violet stain. After fixation (2 min 70% ethanol), dip slide for 30 s into 1% Cresyl violet acetate solution. Remove excess stain on absorbent surface. Dip into 70% of ethanol. Dip into 100% of ethanol. Air-dry briefly for 1 2 min (see Note 2). 4. HE stain. HE staining is used routinely in most histological laboratories and does not interfere with good RNA preparation, if intrinsic RNase activity is low. After fixation, quickly dip slide five to six times in RNase-free distilled water. Stain 1 2 min in Mayer s Hematoxylin. Rinse 1 min in DEPC-treated tap water. Stain 10 s in Eosin Y. Perform a quick increasing ethanol series (70, 96, and 100%). Airdry shortly. 5. Storage. Stained slides can be used immediately or stored at 80 C before LCM (see Note 9). To avoid excess condensation of moisture during thawing, the slides should be frozen in a tightly sealed container (e.g., two slides back to back in a 50 ml Falcon tube) Laser Microdissection: Capture in AdhesiveCaps: Dry Collection 1. An AdhesiveCap (Fig. 2) is filled with a kind of silicon that is completely dry. The intention of AdhesiveCap is to allow LCM without applying any capturing liquid into the caps prior to LCM. Beside the quick relocation of the lifted samples in the cap due to instant immobilization, there is no risk Fig. 2. AdhesiveCap is a collection device filled with an adhesive material. It is especially adapted for a buffer-free sampling of microdissected specimens.

8 124 Burgemeister of evaporation and crystal formation of a capturing buffer during extended specimen harvesting (see Note 10). 2. Other commercially available RNase-free plasticware can be used too (e.g., ABgene #AB-0350; 0.5 ml tubes). If there are no RNase-free tubes available, RNases from regular tubes have to be removed Laser Microdissection: Capture in Routine Caps: Wet Collection When using unfilled regular microfuge tubes, it is recommended to fill a liquid into the cap to improve the adhesion of the captured cells. 1. Pipette 20 ml of lysis buffer into the cap. 2. Perform LCM. 3. The captured cells or cell areas will stick onto the wet inner surface of the cap and will not fall down after the lifting procedure (see Note 7). 4. When using glass-mounted samples (without membrane), it may be advisory to put more liquid (up to 40 ml) into the cap RNA Extraction: Remarks 1. In our hands, the QIAGEN RNeasy FFPE Kit with some specific modifications is most useful. This procedure is very effective and allows a high final concentration of RNA due to a small elution volume. Genomic DNA contamination is minimized by a special DNA removal column (gdna Eliminator spin column). 2. Since normally only stained tissue sections are used for microdissection, the deparaffinization and staining are done according to standard procedures for slides. Furthermore, the incubation with Proteinase K in our protocols is prolonged significantly compared with the QIAGEN protocol, because all our tests with laser microdissected material from various tissues showed higher RNA yields applying extended digestion times. 3. For formalin-fixed samples, a Proteinase K digestion step is essential. The time necessary for optimal Proteinase K digestion depends on many factors such as tissue type, fixation procedure, or element size of lifted material (see Note 11). The RNA solution may be stored at 20 C or used directly for reverse transcription. 4. Quality control by direct analyses such as the Agilent Bioanalyzer is very limited and only possible with large microdissected samples (some 4 mm2 from tissue sections of 5 10 mm thickness). We normally use 5 10 ml of the final RNA solution in an RT reaction of 20 ml using random oligomers (instead of oligot) as primers for the cdna synthesis (see Note 12).

9 Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections RNA Extraction by Using Components of the QIAGEN RNeasy FFPE Kit Add 150 ml of Buffer PKD and 10 ml of Proteinase K to the tube, containing the LCM elements in the AdhesiveCap, and vortex in an upside down position. 2. Use an incubator to digest the samples in an upside down position at 55 C overnight (or for at least 3 h, see Note 11), then vortex and heat at 80 C for 15 min in a heating block. 3. Add 320 ml of Buffer RBC to adjust binding conditions. 4. Mix the lysate thoroughly and transfer it to a gdna Eliminator spin column placed in a 2 ml collection tube. Centrifuge for 30 s at ³8,000 g. Discard the column and save the flow through. 5. Add 720 ml of 100% ethanol to the flow through and mix well by pipetting. Do not centrifuge. Proceed immediately to the next step. 6. Transfer 700 ml of the sample to an RNeasy MinElute spin column placed in a 2 ml collection tube. Close the lid gently and centrifuge for 15 s at ³8,000 g. Discard the flow through. Reuse the collection tube in step Repeat step 6 until the entire sample has passed through the RNeasy MinElute spin column. Reuse the collection tube in step Add 500 ml of Buffer RPE to the RNeasy MinElute spin column. (Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.) Close the lid gently and centrifuge for 15 s at ³8,000 g to wash the spin column membrane. Discard the flow through. Reuse the collection tube in step Add 500 ml of Buffer RPE to the RNeasy MinElute spin column. Close the lid gently and centrifuge for 15 s at ³8,000 g to wash the spin column. After centrifugation carefully remove the spin column from the collection tube so that the column does not contact the flow through. 10. Place the RNeasy MinElute spin column in a new 2 ml collection tube, and discard the old collection tube with the flow through. Open the lid of the spin column and centrifuge at full speed for 5 min. Discard the collection tube with the flow through. It is important to dry the spin column membrane, since residual ethanol may interfere with downstream reactions. 11. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube. Add ml RNase-free water directly to the spin column membrane. Close the lid gently and centrifuge for 1 min at full speed to elute the RNA. The dead volume of the RNeasy MinElute spin column is 2 ml: elution with 14 ml of RNase-free water results in a 12 ml eluate. 12. The RNA solution may be stored at 20 C or used directly for reverse transcription.

10 126 Burgemeister Other RNA Extraction Methods 1. Apart from the QIAGEN Kit, there are many other possibilities and kits to extract RNA from FFPE material. Depending on the material and the experience of the user, even simple procedures such as homemade AGTC methods or Trizol can be quite efficient. 2. If the original extraction protocol does not contain any Proteinase K digestion step, we recommend to apply the following simple procedure. 3. Add 20 ml of digestion buffer containing Proteinase K to the tube containing the LCM elements in the AdhesiveCap. 4. Use an incubator to digest the samples in an upside down position at 55 C overnight. 5. Spin down the lysate in a microcentrifuge (13,400 rcf). 6. Inactivate Proteinase K by heating to 90 C for 10 min. 7. Add the appropriate lysis buffer and mix by intense vortexing; if not proceeding immediately, store the digested samples at 20 or 80 C. 8. Continue with your preferred extraction procedure Quality Control of RNA 1. The most common method used for assessing the integrity of total RNA is to analyze the RNA sample on an agarose gel. In general, at least 200 ng of RNA must be loaded onto the gel. This usually is not practicable with low amounts as usually obtained during laser microdissection. 2. To analyze RNA samples with concentrations down to 50 pg/ml, the Agilent 2100 Bioanalyzer is an alternative to traditional gel-based analysis and provides information about RNA quality (degradation and purity) and quantity. 3. A prognosis of the expected amount of RNA in a tissue is difficult, since many factors such as species, cell/tissue type, fixation, staining, fragmentation, extraction procedure, and others will influence the outcome DNA Extraction 1. Proteinase K digestion step is essential for formalin-fixed samples. The time necessary for optimal Proteinase K digestion depends on many factors such as tissue type, fixation procedure, or element size of lifted material. An overnight digestion (12 18 h) is a good starting point for optimization but shorter digestion times may be tested as well. To our experience at least 3 h digestion should be applied with any extraction procedure and material. 2. Add 15 ml ATL of the QIAamp Micro Kit for the isolation of genomic DNA to the microdissected sample in the AdhesiveCap.

11 Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections Add 10 ml Proteinase K and mix by pulse-vortexing for 15 s. 4. Place the 0.2 ml tube in an upside down position at 56 C in an incubator for 2 18 h by occasional agitation (see Note 13). 5. Add 25 ml Buffer ATL and 50 ml Buffer AL, close the lid and mix by pulse-vortexing for 15 s. To ensure efficient lysis, it is essential that the sample and Buffer AL are thoroughly mixed to a homogeneous solution. 6. Add 50 ml of ethanol (96 100%), close the lid, and mix thoroughly by pulse-vortexing for 15 s. Incubate for 5 min at room temperature (15 25 C). If room temperature exceeds 25 C, cool the ethanol on ice before adding to the tube. 7. Briefly centrifuge the 0.2 ml tube to remove drops from the lid. 8. Carefully transfer the entire lysate to the column without wetting the rim, close the lid, and centrifuge at 6,000 g for 1 min. Place the column in a clean 2 ml collection tube, and discard the collection tube containing the flow through. If the lysate has not completely passed through the column after centrifugation, centrifuge again at a higher speed until the column is empty. 9. Carefully open the column and add 500 ml of Buffer AW1 without wetting the rim. Close the lid and centrifuge at 6,000 g for 1 min. Place the column in a clean 2 ml collection tube, and discard the collection tube containing flow through. 10. Repeat step 9 (see Note 14). 11. Centrifuge at full speed (20,000 g) for 3 min to dry the membrane completely. This step is necessary, since ethanol carryover into the eluate may interfere with some downstream applications. 12. Place the column in a clean 1.5 ml microcentrifuge tube and discard the collection tube containing the flow through. Carefully open the lid of the column and apply 20 ml of distilled water to the center of the membrane. Ensure that distilled water is equilibrated to room temperature (15 25 C). Dispense distilled water onto the center of the membrane to ensure complete elution of bound DNA (see Note 15). 13. Close the lid and incubate at room temperature (15 25 C) for 1 min. Centrifuge at full speed (20,000 g) for 1 min.

12 128 Burgemeister 4. Notes 1. Sometimes lot-to-lot variations in the purchased Cresyl violet powder can lead to weaker staining results if the dye content is below 75%. 2. In most cases, the Cresyl violet staining procedure will be sufficient for cell identification. If an enhancement of the staining intensity is desired, two additional steps of 50% (v/v) ethanol are possible: one step before staining in Cresyl violet, one step after staining in Cresyl violet. Additional intensification can be obtained by increasing the working temperature of all solutions to room temperature. 3. We recommend AdhesiveCap as a collection device for all RNA experiments. After LCM add lysis buffer of your own choice and incubate upside down for 30 min. Subsequently centrifuge the lysate and then apply the further steps of the experiments. 4. DEPC is toxic and should be used under a hood. 5. Always prepare a fresh mixture of capturing buffer and Proteinase K. 6. The detergent Igepal CA-630 in the capturing buffer allows to smear out a small amount of liquid in the whole cap area. 7. Please keep in mind that all kinds of aqueous solutions will run dry during extended working time. 8. It is very important not to use any force to push off the coverslip because this might damage the section. Wait until it falls off by itself. The necessary time depends on the age of the sample and the dryness of the mounting medium and may range from hours to days. Fresh slides (only days old) can be de-coverslipped much faster. From the dry glass slide, sample material can be captured directly by the AutoLPC function of PALM RoboSoftware. 9. For rethawing, the container should not be opened before it is completely warmed up again to ambient temperature. 10. We recommend AdhesiveCap as a collection device for most experiments. After LCM add lysis buffer of your own choice and incubate upside down for 30 min. Subsequently centrifuge the lysate and then apply the further steps of the experiments. 11. The time necessary for optimal Proteinase K digestion depends on many factors such as tissue type, fixation procedure, or element size of lifted material. An overnight digestion (12 18 h) is a good starting point for optimization but shorter digestion times may be tested as well. To our experience at least 3 h

13 Nucleic Acids Extraction from Laser Microdissected FFPE Tissue Sections 129 digestion should be applied with any extraction procedure and material. 12. The use of random or gene-specific primers is important. Reverse transcription of formalin-fixed RNA with standard oligot primers is inefficient and strongly 3 -biased due to the numerous strand breaks and modifications inflicted by the formalin fixation and paraffin-embedding procedure. 13. The time necessary for complete Proteinase K digestion depends on the kind and the amount of collected material. 14. Contact between the column and the flow through should be avoided. Some centrifuge rotors may vibrate upon deceleration, resulting in the flow through, which contains ethanol coming into contact with the column. Take care when removing the column and collection tube from the rotor, so that flow through does not come into contact with the column. 15. QIAamp MinElute Columns provide flexibility in the choice of elution volume. Choose a volume according to the requirements of the downstream application. Remember that the volume of eluate will be up to 5 ml less than the volume of elution solution applied to the column. References 1. Lehmann, U., and Kreipe, H. (2009) Tissue procurement for molecular studies using laserassisted microdissection. Methods Mol Biol. 506, Von Eggeling, F., and Ernst, G. (2007) Microdissected tissue: an underestimated source for biomarker discovery? Biomark Med. 1, Alvarez, H., Corvalan, A., Roa, J.C., Argani P., Murillo F., Edwards J., et al. (2008) Serial analysis of gene expression identifies connective tissue growth factor expression as a prognostic biomarker in gallbladder cancer. Clin Cancer Res. 14, Melle, C., Ernst, G., Schimmel, B., Bleul, A., Koscielny, S., Wiesner, A., et al. (2004) A technical trade for proteomic identification and characterization of cancer biomarkers. Cancer Res. 64, Zhang, Y., Ye, Y., Shen, D., Jiang, K., Zhang, H., Sun, W., et al. (2010) Identification of transgelin-2 as a biomarker of colorectal cancer by laser capture microdissection and quantitative proteome analysis. Cancer Sci. 101, Mori, R., Wang, Q., Danenberg, K.D., Pinski, J.K., and Danenberg, P.V. (2008) Both b-actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer. Prostate. 68, Theophile, K., Jonigk, D., Kreipe, H., and Bock, O. (2008) Amplification of mrna from laser microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR. Diagn Mol Pathol. 17, Schlomm, T., Luebke, A.M., Sultmann, H., Hellwinkel, O.J., Sauer, U., Poustka, A., et al. (2005) Extraction and processing of high quality RNA from impalpable and macroscopically invisible prostate cancer for microarray gene expression analysis. Int J Oncol. 27,