Gene Expression Profiling in Laser-Microdissected Bone Marrow Megakaryocytes

Hussein, Kais

doi:10.1007/978-1-61779-163-5_36

Cited by 4 publications

(3 citation statements)

References 16 publications

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“…Laser microdissection of single cells was performed as described by Hussein (20). In brief, 6 µm cryosections of the reconstituted liver were transferred onto lysine-coated ultraviolet-light-treated membrane slides (MMI) and put on a fluorescence microscope with a SmartCut Plus laser microdissection device (MMI).…”

Section: Methodsmentioning

confidence: 99%

Multiplexing clonality: combining RGB marking and genetic barcoding

Cornils

Thielecke

Hüser

et al. 2014

Nucleic Acids Research

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RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.

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Section: Methodsmentioning

confidence: 99%

Multiplexing clonality: combining RGB marking and genetic barcoding

Cornils

Thielecke

Hüser

et al. 2014

Nucleic Acids Research

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“…71 These, in turn, led to the proposal of molecular classifications for AML 72 and myeloproliferative neoplasms, 73 and, more importantly, to multi-gene panels for mutation assessment in clinical practice. 74 In recent years, further technical improvements (e.g., single-cell analysis, molecular characterisation of laser micro-dissected samples, and gene expression profiling based on RNAsequencing [75][76][77] ) have literally revolutionised BM studies, questioning the value of traditional morphology and phenotypic assessment. 78 Nevertheless, there are many reasons why BM examination is still relevant to our daily clinical practice.…”

Section: A F Ter Gh E DI N I: Th E Evolu Tion Of Bm St U Die S I N Th...mentioning

confidence: 99%

Of drills and bones: Giovanni Ghedini and the origin of bone marrow biopsy

et al. 2022

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Bone marrow (BM) studies are pivotal for the diagnosis of haematological disorders. Their introduction into clinical haematology dates back to the work of GiovanniGhedini , an Italian physician who first conceived BM sampling in 1908.Ghedini's proposal stemmed from his clinical experience and from the scientific developments that characterised his epoch. By presenting selected passages of Ghedini's publications, this report considers the theoretical and historical bases of his work and analyses its practical implications for modern haematology.

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“…Laser capture micro-dissection (LCM) enables the isolation of single cells or small cell groups from histological sections under direct microscopic control [ 1 ]. Combined with quantitative PCR, LCM is a very powerful approach for studying individual transcript expression profiles in discrete cell populations [ 2 ]. Especially for renal biopsies, micro-dissection of its different compartments—glomeruli, tubulointerstitium and arteries/arterioles—is a worthwhile exercise despite its small amounts of degraded RNA.…”

Section: Introductionmentioning

confidence: 99%

Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples

et al. 2018

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BackgroundGlomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve research applications. The major challenge for such studies is to obtain good-quality RNA from small amounts of starting material, as applicable for the analysis of glomerular compartments. In this work, we provide data and recommendations for an optimized workflow of glomerular mRNA analysis.ResultsWith a proper resolution of the camera and screen provided by the next generation of micro-dissection systems, we are able to separate parietal epithelial cells from glomerular tufts. Selected compartment-specific transcripts (WT1 and GLEPP1 for glomerular tuft as well as PAX2 for parietal epithelial cells) seem to be reliable discriminators for these micro-dissected glomerular substructures. Using the phenol–chloroform extraction and hemalaun-stained sections (2 µm), high amounts of Bowman’s capsule transections (> 300) reveal sufficient RNA concentrations (> 300 ng mRNA) for further analysis. For comparison, in unstained sections from a number of 60 glomerular transections upwards, a minimum amount of 157 ng mRNA with a reasonable mRNA purity [A260/A280 ratio of 1.5 (1.4/1.7) median (25th/75th percentiles)] was reversely transcribed into cDNA. Comparing the effect of input RNA (20, 60, 150 and 300 micro-dissected glomerular transections), transcript expression of POLR2A significantly correlated when 60 and 150 laser micro-dissected glomerular transections were used for analysis. There was a lower inter-assay coefficient of variability for ADAMTS13, when at least 60 glomerular transections were used. According to the algorithms of geNormPlus and NormFinder, PGK1 and PPIA are more stable glomerular reference transcripts compared to GUSB, GAPDH, POLR2A, RPLPO, TBP, B2M, ACTB, 18SrRNA and HMBS.ConclusionsOur approach implements compartment-specific glomerular mRNA expression analysis into research applications, even regarding glomerular substructures like parietal epithelial cells. We recommend using of at least 60 micro-dissected unstained glomerular or 300 hemalaun-stained Bowman’s capsule transections to obtain sufficient input mRNA for reproducible results. Hereby, the range of RNA concentrations in 60 micro-dissected glomeruli is low and appropriate normalization of Cq values using our suggested reference transcripts (PGK1 and PPIA) allows compensation with respect to different amounts of RNA purity and quantity.Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0103-x) contains supplementary material, which is available to authorized users.

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Gene Expression Profiling in Laser-Microdissected Bone Marrow Megakaryocytes

Cited by 4 publications

References 16 publications

Multiplexing clonality: combining RGB marking and genetic barcoding

Multiplexing clonality: combining RGB marking and genetic barcoding

Of drills and bones: Giovanni Ghedini and the origin of bone marrow biopsy

Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples

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