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Fanyi Zeng,
Shu-Zhen Huang,
Zhi-Juan Gong,
Mei-Jue Chen,
Don A Baldwin,
Wei Hu,
Hui Qian,
Jing-Bin Yan,
Juan Wang,
Yan Ping Xiao,
Yves Chalandon,
Ashley Ringrose,
Zhao-Rui Ren,
Allen Eaves, Connie Eaves,
Xiaoyan Jiang
Cell Research 04/2013; · 8.19 Impact Factor
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ABSTRACT: Transplantable human hematopoietic stem cells can be routinely detected by their ability to engraft sublethally irradiated
immunodeficient mice with both lymphoid and myeloid progeny for periods of at least 6-8 weeks [1]. These cells include subsets
with both short-term and longterm reconstituting activity, the former being responsible for most of the human cells seen in
fully immunodeficient mice (eg, NOD/SCID-β2microglobulin−/− mice) in the first 8 weeks and the latter being responsible for the lower levels of engraftment seen after the same period
of time in less compromised (eg, NOD/SCID) mice [2]. Interestingly, human cells with short- and longterm hematopoietic reconstituting
potential show further differences in the regulation of their ability to engraft when they are activated into cycle short-term
reconstituting cells being unaffected whereas longterm reconstituting cells lose their transplantability when they transit
S/G2/M [2,3]. A closely related population of primitive human hematopoietic cells (referred to as longterm culture-initiating
cells or LTC-ICs) are detected by virtue of their ability to generate intermediate types of progenitor cells for at least
5 weeks when co-cultured with stromal cell feeder layers, the intermediate progenitors being those that produce colonies of
granulocytes and macrophages and/or erythroblasts in 2-week semi-solid cultures [4]. The proliferative status of these various
stages of primitive human hematopoietic cells is regulated by their exposure to distinct types of both positively acting (mitogenic)
and negatively acting (cytostatic) cytokines. We have recently discovered that stromal-derived growth factor 1 (SDF-1) belongs
to a group of chemokines that act as inhibitors of primitive human progenitor cycling and is unique in its ability to force
the entry of human LTC-ICs into Go. SDF-1 can similarly act on proliferating human cells with longterm hematopoietic reconstituting ability in NOD/SCID mice.
These cells can be shown to be actively proliferating at early times post-transplant in primary NOD/SCID mice by their high
sensitivity to treatment with 5-fluorouracil in vivo or with high specific activity3H-thymidine in vitro as revealed when they are subsequently assayed in secondary NOD/SCID mice. Prior SDF-1 treatment in the
primary mice abrogates this sensitivity. Importantly, this exposure to SDF-1 in vivo also causes a marked increase in the
number of reconstituting cells that can be detected in the secondary mice even without exposure to a cycle-active drug - consistent
with the restoration of engraftment potential by cycling longterm reconstituting stem cells forced to re-enter Go. A similar, albeit less marked effect has also been achieved by SDF-1 treatment of human stem cells stimulated to proliferate
in vitro. These findings demonstrate that cell cycle activation is a major but reversible constraint to the use of proliferating
stem cells for transplantation therapies and suggest a novel approach to overcoming this limitation.
International Journal of Hematology 04/2012; 76:159-159. · 1.27 Impact Factor
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Michael Heuser,
Haiyang Yun,
Tobias Berg,
Eric Yung,
Bob Argiropoulos,
Florian Kuchenbauer,
Gyeongsin Park,
Iyas Hamwi,
Lars Palmqvist,
Courteney K Lai, [......],
Gordon Robertson,
Martin Hirst,
David Kent,
Nicola K Wilson,
Bertie Göttgens, Connie Eaves,
Michael L Cleary,
Marco Marra,
Arnold Ganser,
R Keith Humphries
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ABSTRACT: Pathways defining susceptibility of normal cells to oncogenic transformation may be valuable therapeutic targets. We characterized the cell of origin and its critical pathways in MN1-induced leukemias. Common myeloid (CMP) but not granulocyte-macrophage progenitors (GMP) could be transformed by MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that MN1-leukemogenicity required the MEIS1/AbdB-like HOX-protein complex. ChIP-sequencing identified common target genes of MN1 and MEIS1 and demonstrated identical binding sites for a large proportion of their chromatin targets. Transcriptional repression of MEIS1 targets in established MN1 leukemias demonstrated antileukemic activity. As MN1 relies on but cannot activate expression of MEIS1/AbdB-like HOX proteins, transcriptional activity of these genes determines cellular susceptibility to MN1-induced transformation and may represent a promising therapeutic target.
Cancer cell 07/2011; 20(1):39-52. · 25.29 Impact Factor
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Experimental hematology 05/2011; 39(5):602-3. · 3.11 Impact Factor
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Xiaoyan Jiang,
Donna Forrest,
Franck Nicolini,
Ali Turhan,
Joelle Guilhot,
Calvin Yip,
Tessa Holyoake,
Heather Jorgensen,
Karen Lambie,
Kyi Min Saw,
Emily Pang,
Ranko Vukovic,
Paeta Lehn,
Ashley Ringrose,
Miao Yu,
Ryan R Brinkman,
Clay Smith,
Allen Eaves, Connie Eaves
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ABSTRACT: Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34(+) stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients, we now demonstrate that some, but not all, of these parameters correlate with subsequent clinical response to IM therapy. CD34(+) cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P < .001) and direct sequencing of cloned transcripts from CD34(+) cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders, P < .003). In contrast, CD34(+) cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly, one BCR-ABL mutation (V304D), predicted to destabilize the interaction between p210(BCR-ABL) and IM, was detectable in 14 of 20 patients. T315I mutant CD34(+) cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus, 2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management.
Blood 09/2010; 116(12):2112-21. · 9.90 Impact Factor
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ABSTRACT: Abstract
Background
Expression levels of the cell surface glycoprotein, CD7, and the serine protease, elastase 2 (ELA2), in the leukemic cells of patients with chronic myeloid leukemia (CML) have been associated with clinical outcome. However, little is known about the mechanisms that underlie the variable expression of these genes in the leukemic cells.
Results
To address this question, we compared the level of their expression with the DNA methylation and histone acetylation status of 5' sequences of both genes in leukemic cell lines and primitive (lin<sup>-</sup>CD34<sup>+</sup>) leukemic cells from chronic phase CML patients. DNA methylation of the ELA2 gene promoter did not correlate with its expression pattern in lin<sup>-</sup>CD34<sup>+ </sup>cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus in ELA2 -expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of the CD7 promoter region in the lin<sup>-</sup>CD34<sup>+ </sup>cells from CML patients with high CD7 expression.
Conclusion
These findings indicate a link between epigenetic modifications and CD7 expression in primitive CML cells.
Molecular Cancer. 01/2010;
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ABSTRACT: Human umbilical cord blood (CB) could be an attractive source of hematopoietic repopulating cells for clinical stem cell therapy because of its accessibility and low propensity for unwanted immune reaction against the host. However, CB recipients suffer from severely delayed and often chronically deficient platelet recovery of unknown cause. Here we show that human short-term repopulating cells (STRCs), which predominantly carry early hematopoietic reconstitution after transplantation, display an intrinsically fixed differentiation program in vivo that changes during ontogeny. Compared to adult sources of hematopoietic cells, CB myeloidrestricted STRC-M showed a markedly reduced megakaryocytic and erythroid cell output in the quantitative xenotransplantation of human short-term hematopoiesis in NOD/SCID-beta2m(-/-) mice. This output in vivo was not altered by pre-treating CB cells before transplantation with growth factors that effectively stimulate megakaryocytopoiesis in vitro. Moreover, injecting mice with granulocyte colony-stimulating factor did not affect the differentiation of human STRC. These findings demonstrate that the differentiation capacity of human STRCs is developmentally regulated by mechanisms inaccessible to currently available hematopoietic growth factors, and explain why thrombopoiesis is deficient in clinical CB transplantation.
Stem cells and development 09/2009; 19(5):621-8. · 4.15 Impact Factor
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ABSTRACT: We studied the diagnostic role of CFC assays in myelodysplastic syndromes (MDS) using CFC data from bone marrow (BM) and peripheral blood (PB) of 221 MDS patients, 51 patients with non-malignant causes of cytopenia and/or dysplasia and 50 normal controls. A consistent decrease in BM but not PB multi-lineage and erythroid progenitor frequencies was seen in patients with MDS compared to controls (P<0.05). Automated distinction showed a sensitivity of 87+/-6% and a specificity of 71+/-11% in classifying MDS patients. In conclusion, a defect in early hematopoietic progenitor activity, in particular erythroid activity, distinguishes MDS from non-MDS.
Leukemia research 05/2009; 33(12):1636-42. · 2.36 Impact Factor
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ABSTRACT: Chronic myeloid leukemia (CML) is believed to originate from a normal hematopoietc stem cell acquiring the BCR-ABL fusion gene whose protein product has hyperactive tyrosine kinase activity. Though imatinib mesylate(IM) that targets BCR-ABL kinase activity is now widely used, its curative potential as a single agent is not sure, moreover it is unlikely to eliminate the CML stem cells either, which highlights the necessity to elucidate the molecular mechanism operative in CML stem cells. Previously 16 LongSAGE libraries were established to analyze the CML stem cell and their normal counterparts from various sources. There are numerous novel tags which might represent bona fidetranscripts uniquely expressed in these primitive CML SAGE libraries, which provide us an uniquely opportunity to discovery unknown but important transcripts in these cells. We utilized bioinformatics analyses to sort out these novel tags as the candidates to recover the potential bona fide transcripts, and then with PCR and 3'-RACE (Rapid Amplification of cDNA End) approaches we assessed the validity of them and recovered the 3'-end of the potential novel transcripts originated from these tags with the sequence confirmation. The 5'-RACE is under way to eventually recovery the full-length cDNAs and their gene expression pattern between multiple CML and normal primitive cell samples will be assessed.
12/2008;
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ABSTRACT: Cellular and molecular changes that occur during the genesis of the hematopoietic system and hematopoietic stem cells in the human embryo are mostly inaccessible to study and remain poorly understood. To address this gap we have exploited the human embryonic stem cell (hESC) system to molecularly characterize the global transcriptomes of the two functionally discreet and phenotypically separable populations of multipotent hematopoietic cells that first appear when hESCs are induced to differentiate on OP9 cells.
We prepared long serial analysis of gene expression libraries from lin-CD34+CD43+CD45- and lin-CD34+CD43+CD45+ subsets of primitive hematopoietic cells derived in vitro from hESCs, sequenced them to a depth of 200,000 tags and compared their content with similar libraries prepared from highly purified populations of very primitive human fetal liver and cord blood hematopoietic cells.
Comparison of libraries obtained from hESC-derived lin-CD34+CD43+CD45- and lin-CD34+CD43+CD45+ revealed differences in their expression of genes associated with myeloid development, cellular biosynthetic processes, and cell-cycle regulation. Further comparisons with analogous data for primitive hematopoietic cells isolated from first-trimester human fetal liver and newborn cord blood showed an apparent similarity between the transcriptomes of the most primitive hESC- and in vivo-derived populations, with the main differences involving genes that regulate HSC self-renewal and homing, chromatin remodeling, AP1 transcription complex genes, and noncoding RNAs.
These data suggest that primitive hematopoietic cells are generated from hESCs in vitro by processes similar to those operative during human embryogenesis in vivo, although some differences were also detected.
Experimental Hematology 11/2008; 36(10):1377-89. · 2.90 Impact Factor
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Afshin Raouf,
Yun Zhao,
Karen To,
John Stingl,
Allen Delaney,
Mary Barbara,
Norman Iscove,
Steven Jones,
Steven McKinney,
Joanne Emerman,
Samuel Aparicio,
Marco Marra, Connie Eaves
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ABSTRACT: Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.
Cell stem cell 08/2008; 3(1):109-18. · 23.56 Impact Factor
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ABSTRACT: Understanding the intrinsic pathways that regulate hematopoietic stem cell (HSC) proliferation and self-renewal responses to external signals offers a rational approach to developing improved strategies for HSC expansion for therapeutic applications. Such studies are also likely to reveal new targets for the treatment of human myeloid malignancies because perturbations of the biological processes that control normal HSC self-renewal divisions are believed to drive the propagation of many of these diseases. Here, we review recent findings that point to the importance of using stringent functional criteria to define HSCs as cells with longterm repopulating activity and evidence that activation of the KIT receptor and many downstream effectors serve as major regulators of changing HSC proliferative and self-renewal behavior during development.
Clinical Cancer Research 05/2008; 14(7):1926-30. · 7.74 Impact Factor
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ABSTRACT: Chronic myeloid leukemia (CML) is a clonal multi-step myeloproliferative disease that is initially produced and ultimately sustained by a rare subpopulation of BCR-ABL+ cells with multi-lineage stem cell properties. These BCR-ABL+ CML stem cells are phenotypically similar to normal hematopoietic stem cells which are also maintained throughout the course of the disease at varying levels in different patients. Defining the unique properties of the leukemic stem cells that produce the chronic phase of CML has therefore had to rely heavily on access to samples from rare patients in which the stem cell compartment is dominated by leukemic elements. Here we review past and ongoing approaches using such samples to identify biologically and clinically relevant biomarkers of BCR-ABL+ stem cells that explain their unusual biology and that may help to design, or at least predict, improved treatment responses in CML patients. These studies are of particular interest in light of recent evidence that chronic phase CML stem cells are not only innately resistant to imatinib mesylate and other drugs that target the BCR-ABL oncoprotein, but are also genetically unstable.
Disease markers 01/2008; 24(4-5):201-16. · 1.64 Impact Factor
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ABSTRACT: Suspensions of multipotent hematopoietic stem cells with long-term repopulating activity can now be routinely isolated from adult mouse bone marrow at purities of 30%. A robust method for obtaining these cells in a single step using multiparameter cell sorting to isolate the CD45(mid)lin(-)Rho(-)SP subset is described here, together with a detailed protocol for assessing their regenerative activity in mice transplanted with single cells. These procedures provide unprecedented power and precision for characterizing the molecular and biological properties of cells with hematopoietic stem cell activity at the single cell level.
Current protocols in stem cell biology 12/2007; Chapter 2:Unit 2A.4.
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ABSTRACT: Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However, the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug, a transcriptional repressor, as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin, which resulted in beta-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin-negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression, reexpression of E-cadherin, and suppression of active beta-catenin. Our findings suggest that ligand-induced Notch activation, through the induction of Slug, promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis.
Journal of Experimental Medicine 12/2007; 204(12):2935-48. · 13.85 Impact Factor
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ABSTRACT: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo.
ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice.
Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2, CD3, CD7, CD14, CD16, CD24, CD36, CD38, CD56, CD66b, or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360.
Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that, in combination with other markers, allows easy isolation of the stem cell fraction at unprecedented purities.
Haematologica 10/2007; 92(9):1165-72. · 6.42 Impact Factor
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ABSTRACT: Heterogeneity in the differentiation behavior of hematopoietic stem cells is well documented but poorly understood. To investigate this question at a clonal level, we isolated a subpopulation of adult mouse bone marrow that is highly enriched for multilineage in vivo repopulating cells and transplanted these as single cells, or their short-term clonal progeny generated in vitro, into 352 recipients. Of the mice, 93 showed a donor-derived contribution to the circulating white blood cells for at least 4 months in one of four distinct patterns. Serial transplantation experiments indicated that two of the patterns were associated with extensive self-renewal of the original cell transplanted. However, within 4 days in vitro, the repopulation patterns subsequently obtained in vivo shifted in a clone-specific fashion to those with less myeloid contribution. Thus, primitive hematopoietic cells can maintain distinct repopulation properties upon serial transplantation in vivo, although these properties can also alter rapidly in vitro.
Cell stem cell 09/2007; 1(2):218-29. · 23.56 Impact Factor
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ABSTRACT: Imatinib mesylate treatment causes remissions in a majority of patients with chronic myeloid leukemia (CML), but relapses are an increasing problem. We hypothesized that imatinib-resistant leukemic cells emerge from CML stem cells that acquire BCR-ABL gene mutations even before exposure to BCR-ABL-targeted agents such as imatinib.
Lineage-negative (i.e., immature) CD34+ CD38- CML stem cell-enriched populations were isolated from five patients with chronic phase CML samples by fluorescence-activated cell sorting. To identify BCR-ABL gene mutations, complementary DNAs (cDNAs) prepared from purified CML stem cells were subjected to allele-specific amplification using primers corresponding to 16 kinase domain mutations, with normal bone marrow cells serving as negative controls. We also cloned and directly sequenced BCR-ABL cDNAs prepared from freshly isolated CML stem cells and from their progeny generated after 3-5 weeks of culture.
In 20%-33% of cDNA preparations from freshly isolated CML stem cell-enriched populations, both allele-specific amplification and direct sequencing methods revealed mutations in sequences corresponding to the BCR-ABL kinase domain. Mutations were not observed in cDNA sequences encoding the c-ABL kinase domain that were obtained from similar types of primitive normal cells. More than 70 different BCR-ABL mutations (including frameshift mutations and premature stop codons) were identified in the progeny of cultured CML stem cells. Analysis of individual clones derived from the cultured cells demonstrated that new BCR-ABL mutations were produced.
Primary CML stem cells display instability of the BCR-ABL fusion gene both in vivo and in vitro. Thus, patients may possess leukemic stem cells with BCR-ABL kinase mutations before initiation of BCR-ABL-targeted therapies and would likely be predisposed to develop resistance to these agents.
CancerSpectrum Knowledge Environment 06/2007; 99(9):680-93. · 14.07 Impact Factor
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ABSTRACT: Chronic myeloid leukemia (CML) is sustained by a clonally amplified population of Bcr Abl-positive pluripotent stem cells. Persistence of a large, functionally intact yet suppressed residual normal hematopoietic stem cell population in most patients with CML has made it possible to aim at the development of curative therapies. However, achieving this goal requires the identification of agents that will eradicate the leukemic stem cell population. Several potent Bcr-Abl-targeted drugs have now been introduced into clinical practice with remarkable effects. Nevertheless, accumulating data indicate that the leukemic CML stem cells in patients with chronic phase CML are less responsive to these agents than the bulk of the neoplastic cells. In this article, we review emerging evidence that CML stem cells have a number of unusual properties that underlie their relative insensitivity to treatment, including those that specifically target the Bcr-Abl oncoprotein. The biology of the neoplastic stem cells in patients with CML is clearly important to the future attainment of cures and might also prove a paradigm relevant to other types of malignancies that are sustained by transformed stem cell populations.
Clinical Lymphoma & Myeloma 04/2007; 7 Suppl 2:S71-80. · 1.13 Impact Factor
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Jaswinder Khattra,
Allen D Delaney,
Yongjun Zhao,
Asim Siddiqui,
Jennifer Asano,
Helen McDonald,
Pawan Pandoh,
Noreen Dhalla,
Anna-Liisa Prabhu,
Kevin Ma, [......],
David Charest,
Jeff Stott,
Scott Zuyderduyn,
Richard Varhol, Connie Eaves,
Steven Jones,
Robert Holt,
Martin Hirst,
Pamela A Hoodless,
Marco A Marra
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ABSTRACT: We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.
Genome Research 01/2007; 17(1):108-16. · 13.61 Impact Factor
