Workshop Outline. Laser Capture Microdissection=molecular analysis of specific cells. LCM sample is a genomic sample.

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Eugenia Walker
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1 Workshop Outline LCM sample is a genomic sample. Clinical vs experimental LCM sample. Genomic sample collection and stabilization. LCM sample preparation: microtomy, cryotomy, staining. Start-up QC of LCM sample. Storage of LCM sample. HANG ON- IT S GOING TO BE AN INTENSE 45 MINUTES! Disclaimers Laser Capture Microdissection=molecular analysis of specific cells Reference to any specific commercial products, processes, manufacturers or company does not constitute endorsement or recommendation by the U.S. Government (the National Cancer Institute and the National Institute of Environmental Health Sciences) or SAIC Frederick. NIH/NCI/NIEHS and SAIC Frederick are not responsible for any equipment, software or related technical problems demonstrated in this presentation. This presentation is intended for educational purposes only. Funded by NCI Contract HHSN E Tissue Specific cells Tissue section Cell lysate RNA DNA Protein

2 Molecular analysis of LCM sample Mutation Analysis RNA, DNA Protein retrieved from LCM sample Gene Expression Microarray Start Fresh and Sterile Enzymes, naturally found in tissue, are reactivated unless desiccated and work during LCM to destroy your protein targets (including DNA and RNA). This is a BAD thing if you are looking for DNA, RNA or other protein information! Real-Time PCR Gene Expression Analysis Ciphergen Proteomics Protein Chip/Mass Spec Slide thanks to Molecular Devices (modified) Proteomic 2D Gels Time, temperature and water are three factors to control when handling samples for LCM and analyzing for molecular data. Fresher and Faster!!!!!!! LCM sample=genomic Sample Clinical and Experimental LCM Sample Collection necropsy biopsy Stabilization fixation freezing Preparation for analysis OCT block (fresh or fixed tissue) Paraffin block (fixed tissue) processing and embedding LCM slide preparation and dissection Extraction (RNA,DNA,proteins) Intact RNA, DNA, Proteins are crucial for valid molecular analysis. Take care of their integrity during all the steps. Clinical sample (human biopsy) Preanalytical variability is difficult to control. Main focus on sample preparation for retrieval, and analysis of compromised nucleic acids and proteins in the sample Experimental sample (model animals) Standard conditions and methods to minimize preanalytical variability in gene expression Main focus on sample collection and preparation to preserve integrity of intact nucleic acids and proteins in the sample

Preservation of RNA Integrity in Target Organs During Necropsy. RNAse-free set-up. Separate instruments for separate tissues. RNAse-free fixative. Priority of tissue removal based on RNA stability.

lung, thymus, heart and heart atria, trachea, esophagus, pituitary and thyroid/parathyroid complex) for RNA retrieval.

Dissector #2 Necropsy: lung, thymus, heart, trachea, esophagus with a lower jaw in block head, spleen, kidney, reproductive fat, ovaries and bladder Distribution: tissues for sub-dissection.

3 Preservation of RNA Integrity in Target Organs During Necropsy. RNAse-free set-up. Separate instruments for separate tissues. RNAse-free fixative. Priority of tissue removal based on RNA stability. Six Minute Procedure for Multiple Tissue Harvest (female) Study objective: collection of 15 tissues (xiphoid process, spleen, kidney's cortex and medulla layers, reproductive fat, ovaries, bladder, lung, thymus, heart and heart atria, trachea, esophagus, pituitary and thyroid/parathyroid complex) for RNA retrieval. Dissector #1 Euthanizes animals Cuts tissues in RNAlater following its dissection Assists the needs of the dissector #2. Dissector #2 Necropsy: lung, thymus, heart, trachea, esophagus with a lower jaw in block head, spleen, kidney, reproductive fat, ovaries and bladder Distribution: tissues for sub-dissection. Dissector #3 Separates the lower jaw and, passes it to dissector #4 Necropsy: esophagus, heart, atria, trachea and lung Dissector #6 Stabilization of dissected tissues (freezing, fixation or incubation in RNA-later) within 6 minutes after euthanasia. Supervises the dissections through out the entire necropsy session (time tracking, necropsy documentation, etc.) Cleans ovaries, xiphoid process, and bladder from contaminating tissue under dissecting microscope. Dissector #5 Removes pituitary out of the head Sub-dissects kidney Dissector #4 Separates; thyroid/parathyroid complex off the trachea. Targets are different; tailor necropsy protocol to molecular endpoint Stability of RNA in organs of 10 week old C57BL/6J Mice Tissue Brain (whole) Liver Lung Prostate Parotid Pancreas RNA stability stable unstable very unstable extremely unstable 15 after euthanasia Mouse liver Mouse prostate Fulfilling the Requirements: Frozen vs. Fixed Sample Requirements Fixed sample Frozen sample Morphology excellent Depends on a tissue type (from very good to poor) Suitability for IHC Depends on fixation type and conditions suitable DNA RNA Proteins In-situ hybridization Laser Capture Microdissection Degradation level depends on fixation, processing and storage Could be chemically altered Often significantly degraded. Degradation level depends on fixation, processing and storage and tissue type. Cross-linked, need specific retrieval Suitable for some methods of protein analysis. Depends on a fixative and RNA integrity in a section. Good to poor rate of success. DNA is suitable for some RNA is significantly degraded, suitable for some. Low yield. Good to poor rate of success. downstream downstream methods of protein analysis High rate of success Slightly degraded, suitable for all

Optimal trim <= 3mm Sterile Blades New bottle of OCT freezing media.

Slides with frozen sections kept in dry ice while cutting.

Freshly prepared fixatives Low temperature during fixation if possible RNAse-free reagents

5-2 hr-modified Carnoy s, 12-24 hr- 10% NBF) on a shaker. NIH/NIEHS website: www.niehs.nih.

RNAlater is a saturated salt solution that rapidly permeates fresh tissue and protects RNA

4 Fresh frozen sample is optimal. Preservation of RNA Integrity in Target Organs During Fixation. Optimal trim <= 3mm Sterile Blades New bottle of OCT freezing media. Sterilized forceps, blades, molds (RNA). Metal plate on dry ice for even freezing. Slides with frozen sections kept in dry ice while cutting. Store in -80ºC ultra-freezer and use within one week. Freshly prepared fixatives Low temperature during fixation if possible RNAse-free reagents and conditions Short fixation (4-6 hr 4%PFA, hr-modified Carnoy s, hr- 10% NBF) on a shaker. NIH/NIEHS website: RNAlater as RNA Stabilizer. RNAlater is a saturated salt solution that rapidly permeates fresh tissue and protects RNA from degradation. Fresh frozen human kidney biopsy after LCM RNAlater preserved biopsy after LCM Automated Processor Short Cycle for Genomic Sample 4% PFA Modified Carnoy s 85% ETOH 10, RT 95% ETOH 15, RT 95% ETOH 15, RT 100% ETOH 10, RT Xylene 20, RT Xylene 20, RT,on a shaker, move into a processor Xylene Xylene 20, RT 20, RT

RNA Quality from Paraffin Blocks RIN=3.1 RIN=8.

Carnoy s 3. 4% Paraformaldehyde 1. Flat Section Beneath the Anti-Roll Plate 2.

NIEHS Whole Lysis Procedure RNAse-free conditions: - Wipe everything (including brushes,

with 100% alcohol. - Install a new disposable blade for each sample.

Facing the block, discard 5 first sections.

5 RNA Quality from Paraffin Blocks RIN=3.1 RIN= Mounting of OCT Section on PET Slide RIN= Archival Sample 10% NBF 2. Carnoy s 3. 4% Paraformaldehyde 1. Flat Section Beneath the Anti-Roll Plate 2. Anti-roll plate removed, section should be detached from the knife and attached to the metal plate by gentle pressure. 3. Cool a UV- treated PET slide and immediately pick up the section by the membrane window, touching the membrane with your finger. Preservation of RNA Integrity during microtomy and cryomicrotomy. NIEHS Whole Lysis Procedure RNAse-free conditions: - Wipe everything (including brushes, inside surface of the chamber and outer surfaces of the cryostat, and microtome surfaces) with 100% alcohol. - Install a new disposable blade for each sample. - RNAse-free water bath, individual container for block soaking. Facing the block, discard 5 first sections. Use anti-roll plate for cryo sections and automated microtome for paraffin sections. Move cryo sections on dry ice immediately after cutting (don t dry). Dry paraffin sections at +56 C for 2 hours, cool at room temperature, and. Transfer to -80 C storage until use. 1. Pipette 50 µl of lysis buffer on to sample. 2. Wait 30 seconds. 3. Pipette buffer back into tip. 4. Dispense into a µl microtube

Is RNA good for the desired downstream? Evaluation of an LCM staining protocol RNA integrity (RIN) before and after LCM. Sample Frozen (OCT) LCM block cells Mouse ileum 10 8.5 Mouse ileum 10 8.

4 6.7 Human colorectal 9.2 7.0 biopsy Human skin 9.5 6.3 biopsy Human skin 9.4 6.7 biopsy Human skin 7.5 5.3 RIN 8 RIN 7 RIN 5 any application microarray RT-PCR Q-PCR nanostring 1.

Spin down before use, pipette from surface, don t pick the last 50 ul from the tube) - 10. 5. 100% ETOH to rinse stain off (needs to be changed after 3 slides), flip gently once. 6.

6 Is RNA good for the desired downstream? Evaluation of an LCM staining protocol RNA integrity (RIN) before and after LCM. Sample Frozen (OCT) LCM block cells Mouse ileum Mouse ileum Mouse ileum Mouse ileum Mouse ileum Mouse ileum Mouse ileum Mouse ileum Mouse ileum Mouse ileum Mouse stomach Mouse stomach Human colorectal biopsy Human skin biopsy Human skin biopsy Human skin RIN 8 RIN 7 RIN 5 any application microarray RT-PCR Q-PCR nanostring % ETOH (100 ml): acetic acid (3 ml) 20C, 30 sec 2. methyl green (vector) ul + 2 ul RNase inhibitor- 40, drain slide on the kimwipe. 3. methyl green (vector) ul + 2 ul RNase inhibitor- 40, drain slide on the kimwipe. 4. Apply 100 ul of cresyl violet/ eosin mix (300 cresyl+100 eosin+ 400 nuclease free water. Spin down before use, pipette from surface, don t pick the last 50 ul from the tube) % ETOH to rinse stain off (needs to be changed after 3 slides), flip gently once % ETOH-RT, 30 sec % ETOH-RT, 30 seconds 8. Xylene-RT, flip once, 2 min, 9. Xylene-RT, 3 min, 10. Airdry in the hood- 5 min RNA extracted from the frozen section RIN = 9.5 Scrap from the stained slide RIN=7.7 Critical Points of the LCM Staining Protocol Exact timing of staining steps. Water- push it out of the protocol! Stain not more then 4-6 slides at a time. Slide storage in xylene (up to 2 hours) and in a desiccator (up to 1 hour). Storage and RNA Integrity. RIN of different mouse tissue RNA samples before and after 3 year storage RNA stability in storage depends on tissue type and initial RNA quality Tissue RIN before storage Brain Lung Parotid Kidney Bladder Stomach Small intestine Pancreas Bone Liver Esophagus Atria Adrenal Eye RIN after storage

7 Storage of Blocks. Paraffin blocks. After completion of sectioning, wrap the block in Parafilm, place in air tight bag or food saver bag, seal and return to the storage. Store blocks at RT or +4ºC for DNA and at -20ºC for RNA. OCT (frozen) blocks. Store blocks at -80ºC at the back of the freezer. Handle and transfer blocks to -80ºC storage on dry ice. Minimize block time inside the cryostat during cutting. Conclusions Treat LCM sample as a genomic sample. Take care of LCM sample molecular integrity at all steps of sample collection and preparation. QC sample preparation for the best possible result. Think- success! -20ºC Storage of Slides Paraffin section of mouse embryo RT Acknowledgements SAIC, NCI Frederick Dr. Serguei Kozlov Dr. Keith Collins Staff of the Pathology-Histotechnology Laboratory Store OCT slides at -80 C at the back of the freezer. Cut slides for molecular right before use for the best results. NIEHS Dr. Robert Sills Julie Foley

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Downloaded from and current as of 02/01/2008 Downloaded from http://www.moleculardevices.com and current as of 02/01/2008 www.arctur.com Tissue Scrape Protocol for Verifying RNA Quality Using the PicoPure RNA Isolation Kits Introduction Arcturus