Arcturus® Laser Capture Microdissection (LCM) Web Seminar Series
Click on the desired link below to listen to and view recordings of past Arcturus Web Seminars.
Remember to check the Molecular Devices website for details on upcoming Arcturus LCM events.
Recorded Web Seminars
Event Date
Web Seminar Details
December 5, 2008
Gene expression analysis in homogenous single cell populations in the superior temporal gyrus in postmortem brains from subjects with schizophrenia
Charmaine Pietersen, Ph.D., Laboratory for Structural and Molecular Neuroscience, McLean Hospital.
ABSTRACT: For over 20 years it has been postulated that disturbances of the postnatal developmental synaptic pruning process may contribute to the onset of schizophrenia, with symptoms persisting throughout the patients lifetime. In support of this hypothesis, recent postmortem studies have revealed that deficits of glutamatergic synapses may in fact be a prominent pathophysiologic feature of the illness. However, the possible molecular mechanisms that underlie the postulated disturbances of synaptic pruning have been largely unexplored. Here, we hypothesize that disturbed brain-derived neurotrophic factor (BDNF) signaling via its high-affinity receptor trkB may contribute to the onset of schizophrenia via at least 2 mechanisms. First, the functional maturation of the subset of inhibitory interneurons that contain parvalbumin (PV), which is critically dependent upon intact BDNF/trkB signaling, will be delayed. This will lead to excessive loss of synapses by prolonging the developmental synaptic pruning process. Second, disturbed BDNF/trkB signaling will compromise the glutamatergic innervation of PV neurons and, via a disinhibitory mechanism, this will render the downstream pyramidal neurons more susceptible to excitotoxicity, resulting in excessive loss of synapses. These hypothesized events predict specific cellular and molecular signatures that define the endpoint of disease progression and can be studied in postmortem brains from patients in the chronic phase of the illness. Hence, in a cohort of postmortem brains from 10 schizophrenia and 10 matched normal control subjects, we use the Arcturus laser capture microdissection system to isolate subsets of pyramidal and inhibitory neurons in the superior temporal gyrus (BA 42), which is among the cortical regions that exhibit the most pronounced gray matter reduction during the early course of schizophrenia. We routinely obtain between 28.9-60.3µg of aRNA after two rounds of T7-based linear amplification from approximately 500 captured neurons. Expression profiling and real-time polymerase chain reaction experiments are in progress to examine how BDNF/trkB and glutamatergic signaling may be altered in a cell type-specific manner. Findings of this study may inspire the conceptualization of novel therapeutic and preventive strategy that targets the pathophysiologic process of disease progression of schizophrenia.
Applying Paradise®PLUS Reagent System for the Molecular Classification of Cancers of Uncertain Primary Origin
Mark Erlander, Ph.D., Chief Scientific Officer, AviaraDX, Inc.
ABSTRACT: For genomic testing of solid tumors to have an impact on patient management, the test needs to be not only medically useful but also compatible with formalin-fixed paraffin-embedded tissue preservation. Currently in the U.S., the majority of tumor biopsies are preserved by formalin-fixation followed by paraffin embedding (FFPE) to maintain the cyto-architecture of the tissue. It is well known that formalin-fixation of tissue causes RNA to be cross-linked to proteins via covalent addition of monomethylol groups to nucleic acid bases. This cross-linking not only hampers the ability to extract RNA but also decreases the conversion of mRNA to cDNA template by inhibiting the processivity of reverse transcriptase by these methylol modifications. At AviaraDx, we are a high complexity CLIA laboratory, and we have circumvented these technical issues by using Paradise® Plus. I will present here a specific application for the use of Paradise® Plus in which we have developed and validated a laboratory-developed test for the classification of cancers for which the primary origin is uncertain.
Measurement of Gene Expression in Formalin Fixed and Paraffin Embedded (FFPE) Tissue: A Comparison of Different Technologies for RNA Isolation and Reverse Transcription
Michelle Davey, Research Associate, Atlantic Cancer Research Institute, Moncton , New Brunswick Canada
ABSTRACT: Summary of our findings related to the study we conducted that compared commercially available total RNA isolation methods for quantity and quality of RNA extracted from serial breast tissue FFPE sections. We also evaluated methods for reverse transcription of the recovered RNA. These experiments were performed as part of a larger study towards the selection of methods for accurate, reproducible, quantitative analysis of specific RNA levels in standard FFPE tissue specimens. The results provided us with a method that would allow our laboratory and others to take a giant step towards unlocking the previously inaccessible wealth of information in FFPE tissue archives.
The quantitative determination of gene expression levels is a powerful approach for comparative analysis of normal and neoplastic tissues and can yield valuable information towards the discovery of disease mechanisms and identification of diagnostic, prognostic and/or therapeutic cancer targets. Although sensitive and specific methods for studying RNA derived from fresh and frozen tissues are well described, the use of such tissues is restricted by their limited availability and logistical impracticalities. Furthermore, while the development of techniques such as cDNA array hybridization and real-time PCR make mRNA quantisation possible, proper interpretation of the clinical significance of the results requires examination of large numbers of tissue specimens representing different disease stages, tumor types and grades, and outcomes.
Formalin fixed paraffin-embedded (FFPE) tissues represent a large invaluable resource of well-characterized retrospective specimens whose histopathology is well-preserved and for which long-term clinical follow-up data exists. Until recently, RNA processing from tissue fixed in formalin was considered unworkable since the amount of recoverable RNA is so small, the extracted RNA is usually extensively degraded, and the RNA is chemically modified. The development of modified RNA extraction and reverse transcription procedures specifically designed for FFPE tissues, the advent of novel RNA amplification methods, and the introduction of highly sensitive quantitative real-time PCR procedures now open up the possibilities for rapid and specific quantisation of minute amounts of fragmented RNA obtained from FFPE tissues.
Virginia Espina, MS, MT(ASCP), Research Asst Professor, George Mason University , Center for Applied Proteomics and Molecular Medicine
ABSTRACT: The combination of laser capture microdissection protocols with clinical proteomic applications is the new frontier of translational medicine. While genomics provides information regarding the underlying somatic mutations that can lead to cancer and other diseases, proteomic technologies now exist that are able to elucidate protein-protein interactions, quantify post-translational modifications, and map cellular kinase pathways. The ability to monitor the in vivo state of cell signaling proteins before and after treatment will potentially augment the ability to design individualized therapy regimens via the mapping of aberrant cell signaling phenotypes. The mapping of these protein pathways will further the development of rational drug targets. Low abundance analytes such as phosphorylated and non phosphorylated proteins, in specimens consisting of a few thousand cells, can be quantified with reverse phase protein microarray technology.
This presentation will emphasize technical aspects of LCM in relation to downstream proteomic protocols. Breast cancer, Non-small cell lung cancer and childhood Rhabdomyosarcoma will be discussed in the context of microdissection for individualized therapy.
Breast Cancer: Core needle biopsies of breast tissue were obtained for women enrolled in a multi-center, SPORE trial for neo-adjuvant treatment coupled with imaging. Frozen tissue sections were microdissected from 3 sections/patient for the pre-surgery sample. Cellular yield varied from 300 – 3,000 cells/sample. Samples with >75% tumor were selected for microdissection and whole slide tissue lysis as a comparison of protein signatures in microdissected cell populations versus heterogeneous tissue lysates.
Non-small Cell Lung Cancer : Only 10-19% of Non-small cell lung carcinoma (NSCLC) patients respond to gefitinib treatment. This subset of patients has been associated with mutations in the ATP- binding pocket of the tyrosine kinase domain of the Epidermal Growth Factor Receptor (EGFR/erbB1). Several possible molecular explanations include: 1) tumors responding to gefitinib must be driven by EGFR signaling, and specifically will have the receptor phosphorylated and activated, 2) non-responders have an alternative pathway activated, or 3) EGFR is interconnected in all types of lung cancer. Reverse phase protein microarrays provide a means of addressing these questions with a sensitivity in the femtomolar range with a CV of 10%. 27 frozen, human lung adenocarcinoma surgical specimens were microdissected for monitoring the state of the erbB1 (EGFR) receptor driven signaling cascade. The reverse phase protein microarrays recapitulated both the phosphorylation state of the erbB1 receptor as well as a large portion of the downstream cascade.
Rhabdomyosarcoma: We combined laser capture microdissection with multiplexed phosphoproteomic analysis using reverse phase protein microarray technology to identify a molecular signature of RMS tumors that responded to therapy. The in vivo state of multiple kinase driven signal pathways was evaluated with a panel of thirteen specific antibodies developed based upon on-going molecular profiling work suggesting that subsets of cell signaling proteins were contributing to biological behavior. Characterization of molecular signatures for RMS tumors serves two purposes 1) stratifying RMS patients for therapy and 2) discovery of new therapeutic regimens for improving treatment outcomes.
Practical Applications of Laser Capture Microdissection (LCM)
Anna Pecherskaya, MS, LCM Facility Manager, Fox Chase Cancer Center
ABSTRACT : Laser capture microdissection (LCM) is the procedure for procuring pure cell population from the specific microscopic regions of tissue sections and can facilitate comparative genomics studies between different cell populations from the same individual. Different aspects of sample collection, tissue fixation and staining can affect the quality of RNA isolated from LCM material. Therefore, establishment of reliable protocols and quality control steps for both frozen and formalin-fixed paraffin-embedded (FFPE) tissues is important. Bioanalyzer profiles are a reliable means to assess RNA quality from frozen tissue. Functional RT-PCR assays using a panel of gene-specific primers provide a more reliable indication of the quality of RNA isolated from FFPE tissues in downstream analysis. Bioanalyzer profiles and agarose gels are used to control the quality of amplified material. NanoDrop analysis is used to measure dye incorporation before processing to microarray hybridization. The success of the dye incorporation as well as the strength of the signal on microarray step substantially depends on the quality of the aRNA. The usefulness of the qualitative and quantitative analysis of amplification performance and following labeling is demonstrated for the different RNA profiles. Using these protocols we show practical approaches of the LCM technology and that it is possible to obtain reliable microarray data from FFPE samples collected from clinical resection specimens. The Arcturus Paradise Reagent System, which allows robust RNA isolation and amplification using very low RNA input, and custom-designed 22,000 oligonucleotide arrays (Agilent) were used in the present study. An in-depth comparison of microarray data generated from FFPE human colon adenocarcinoma tissue with that obtained from frozen matched specimens was accomplished and high correlation coefficients for the gene expression profiles were obtained.
Validation of LCM/qRT-PCR to Profile the Blood-Brain Barrier Transcriptome of the Cerebral Microvasculature In Situ
Dr. Joel Pachter, Professor of Cell Biology and Director of Blood-Brain Barrier Laboratory, University of Connecticut Health Center
ABSTRACT: Laser capture microdissection (LCM) provides a novel means to selectively retrieve brain endothelial cells from their microvascular branches in situ , and is amenable to downstream applications such as quantitative, real-time, polymerase chain reaction (qRT-PCR) for expression profiling of genes associated with BBB function (BBB-associated genes). Owing to its extreme complexity, however, this technique must first be thoroughly validated to determine that it is highly reproducible: specifically, that it does not suffer from technical variability (associated with adjacent tissue sections from the same animal) or biological variability (associated with sections from different animals). Furthermore, novel efforts to minimize endothelial contamination by undesired cell types (e.g., smooth muscle cells) need be undertaken. To determine whether there is detectable technical variability, brain capillary endothelial cells from a restricted brain area were retrieved by LCM, using an Arcturus PixCell IIe, from consecutive serial sections of one mouse brain, and were pooled into two samples. qRT-PCR-generated expression profiles of sixteen BBB-associated genes were then compared for inter-sample fidelity. To additionally evaluate biological variability, this procedure was repeated on tissue obtained from different animals. A linear correlation (R 2 = 0.99) was observed between different sections from the same animal, which was confirmed by Kendall-Rank Order Correlation Test at p<0.05, and signified that there was no significant technical variability amongst samples. Furthermore, 3-D linear correlation in x, y, and z planes was seen when gene profiling was conducted on sections from three different animals, and two-way ANOVA confirmed the lack of biological variability. Lastly, a combined UV/IR laser approach using the Arcturus XT was employed to accomplish a more selective retrieval of endothelial cells from arterioles. Upon UV-mediated ‘burning' of perivascular smooth muscle cells, followed by IR-mediated ‘lifting' of endothelial cells, the endothelial marker CD-31 was significantly enriched – though at the expense of some RNA loss. Collectively, these findings argue that coupling LCM with qRT-PCR provides a reproducible and proficient means to profile BBB-associated gene expression patterns in highly-enriched populations of CNS microvascular endothelial cells.
