Laser Capture Microdissection Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR Preparation Software (PREXCEL-Q).
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ABSTRACT:
SUBMITTER: Sow FB
PROVIDER: S-EPMC2885795 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data.ResultsThe protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater® Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number (RIN). RNA was then enriched using the TruSeq® RNA Access Library Prep Kit (Illumina®) and sequenced on HiSeq® 2500 platform (Illumina®). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis.ConclusionsThe described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.
| S-EPMC6009967 | biostudies-literature
Project description:We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.
| S-EPMC4215031 | biostudies-literature
Project description:Dormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state-of-the-art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up- or down-regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a primary experimentally-derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence.
| S-EPMC5516136 | biostudies-literature
Project description:Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.
| S-EPMC6971024 | biostudies-literature
Project description:Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus(XT) instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.
| S-EPMC3766583 | biostudies-literature
Project description:The goal of the study was to characterize the whole genome transcriptome profiles of pure sample population of human ameloblastoma epithelial cells for examination of molecular pathways.
2016-09-01 | GSE68531 | GEO
Project description:BackgroundLaser capture microdissection (LCM) has successfully isolated pure cell populations from tissue sections and the combination of LCM with standard genomic and proteomic methods has revolutionized molecular analysis of complex tissue. However, the quantity and quality of material recovered after LCM is often still limited for analysis by using whole genomic and proteomic approaches. To procure high quality and quantity of RNA after LCM, we optimized the procedures on tissue preparations and applied the approach for cell type-specific miRNA expression profiling in colorectal tumors.ResultsWe found that the ethanol fixation of tissue sections for 2 hours had the maximum improvement of RNA quality (1.8 fold, p = 0.0014) and quantity (1.5 fold, p = 0.066). Overall, the quality (RNA integrity number, RIN) for the microdissected colorectal tissues was 5.2 +/- 1.5 (average +/- SD) for normal (n = 43), 5.7 +/- 1.1 for adenomas (n = 14) and 7.2 +/- 1.2 for carcinomas (n = 44). We then compared miRNA expression profiles of 18 colorectal tissues (6 normal, 6 adenomas and 6 carcinomas) between LCM selected epithelial cells versus stromal cells using Agilent miRNA microarrays. We identified 51 differentially expressed miRNAs (p <= 0.001) between these two cell types. We found that the miRNAs in the epithelial cells could differentiate adenomas from normal and carcinomas. However, the miRNAs in the stromal and mixed cells could not separate adenomas from normal tissues. Finally, we applied quantitative RT-PCR to cross-verify the expression patterns of 7 different miRNAs using 8 LCM-selected epithelial cells and found the excellent correlation of the fold changes between the two platforms (R = 0.996).ConclusionsOur study demonstrates the feasibility and potential power of discovering cell type-specific miRNA biomarkers in complex tissue using combination of LCM with genome-wide miRNA analysis.
| S-EPMC2853520 | biostudies-literature
Project description:BACKGROUND: Previous studies of brain and peripheral tissues in schizophrenia patients have indicated impaired energy supply to the brain. A number of studies have also demonstrated dysfunction of the microvasculature in schizophrenia patients. Together these findings are consistent with a hypothesis of blood-brain barrier dysfunction in schizophrenia. In this study, we have investigated the cerebral vascular endothelium of schizophrenia patients at the level of transcriptomics. METHODOLOGY/PRINCIPAL FINDINGS: We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from schizophrenia patients and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using two independent microarray platforms, Affymetrix HG133plus2.0 GeneChips and CodeLink Whole Human Genome arrays. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. We then compared neuronal and endothelial data separately between schizophrenic subjects and controls. Analysis of the endothelial samples showed differences in gene expression between schizophrenics and controls which were reproducible in a second microarray platform. Functional profiling revealed that these changes were primarily found in genes relating to inflammatory processes. CONCLUSIONS/SIGNIFICANCE: This study provides preliminary evidence of molecular alterations of the cerebral microvasculature in schizophrenia patients, suggestive of a hypo-inflammatory state in this tissue type. Further investigation of the blood-brain barrier in schizophrenia is warranted.
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Project description:Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their coexistence with stem cells, making it difficult to culture Paneth cells alone in vitro. Using a simplified toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region, and surfactant-assisted one-pot protein digestion, we identified more than 1300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes, and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues: a comparable proteomic coverage can be achieved with 3600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplified workflow enables profiling of Paneth cell-associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.
| S-EPMC9671084 | biostudies-literature
Project description:Laser capture microdissection (LCM) enables precise dissection and collection of individual cell types from complex tissues. When applied to plant cells, and especially to woody tissues, LCM requires extensive optimization to overcome such factors as rigid cell walls, large central vacuoles, intercellular spaces, and technical issues with thickness and flatness of the sections. Here we present an optimized protocol for the laser-assisted microdissection of developing xylem from mature trees: a gymnosperm (Norway spruce, Picea abies) and an angiosperm (aspen, Populus tremula) tree. Different cell types of spruce and aspen wood (i.e., ray cells, tracheary elements, and fibers) were successfully microdissected from tangential, cross and radial cryosections of the current year's growth ring. Two approaches were applied to achieve satisfactory flatness and anatomical integrity of the spruce and aspen specimens. The commonly used membrane slides were ineffective as a mounting surface for the wood cryosections. Instead, in the present protocol we use glass slides, and introduce a glass slide sandwich assembly for the preparation of aspen sections. To ascertain that not only the anatomical integrity of the plant tissue, but also the molecular features were not compromised during the whole LCM procedure, good quality total RNA could be extracted from the microdissected cells. This showed the efficiency of the protocol and established that our methodology can be integrated in transcriptome analyses to elucidate cell-specific molecular events regulating wood formation in trees.
| S-EPMC5209384 | biostudies-literature