High-dimensional transcriptional analyses of UC Colon comparing RNALater to DMSO collection method
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE108746 | GEO | 2019/01/18
REPOSITORIES: GEO
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Project description:Recently, combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomics changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.
2019-07-25 | GSE127248 | GEO
Project description:Recently, combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomics changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.
2019-07-25 | GSE127246 | GEO
Project description:In this study we performed comprehensive analysis of changes in cell survival, vital parameters, plasticity, as well as transgene expression of placental MSCs after temperature fluctuations within the liquid nitrogen steam storage, mimicking long-term preservation in practical biobanking, transportation, and temporal storage. It was shown that viability and metabolic parameters of placental MSCs did not significantly differ after temperature fluctuations in the range from -196ºC to -100ºC in less than 20 cycles in comparison to constant temperature storage. However, increasing of the temperature range to -80ºC as well as increasing the number of cycles, leads to significant lowering of these parameters after thawing. The number of apoptotic changes increases depending on the number of cycles of temperature fluctuations. Besides, adhesive properties of the cells after thawing are significantly compromised in the samples subjected to temperature fluctuations during storage. Plasticity of placental MSCs was not compromised neither after cryopreservation with constant end temperatures nor with temperature fluctuations. However, regulation of various genes after cryopreservation procedures significantly varies. Alterations in structural and functional parameters of placental MSCs after long-term preservation should be considered in practical biobanking due to potential temperature fluctuations in samples. At the same time, plasticity and transgene expression are not compromised during studied storage conditions, while certain gene regulation is observed.
2017-03-17 | GSE85097 | GEO
Project description:Recombinant human erythropoietin administration studies involving transcriptomic approaches have demonstrated a gene-expression signature that could aid detection of blood doping. However, current anti-doping testing does not involve blood collection into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood leftover from standard haematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservative. Whole blood samples were collected from thirteen and fourteen healthy males, for long-term and short-term storage experiments. Long-term storage: whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., −80°C) storage and RNA extracted. After storage, K2EDTA tubes were thawed and extracted using GeneJET RNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania) or Tempus™ Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA). RNA quality and purity was sufficient for gene expression analysis. Principle Component Analysis of microarray and RNA-seq gene expression data for long-term storage: When comparing gene expression between blood tubes with and without RNA preservation, 6% (4058 transcripts) were differentially expressed. RNA quantity, purity and integrity was not significantly compromised from long-term storage in blood storage tubes lacking RNA preservative, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.
2021-08-13 | GSE181970 | GEO
Project description:We developed and validated a simple method for viable cryopreservation of whole blood, without any preprocessing, Simple prEservatioN of Single cElls (SENSE), with granulocyte depletion for generating high-quality single-cell profiles. We performed rigorous in-depth characterization of the SENSE method on clinical blood samples and compared it to the conventional multistep density-gradient isolation of peripheral blood mononuclear cells (PBMCs) method. The SENSE method is an effective and simple solution for the cryopreservation of blood samples in clinics/labs and single cell profiles generation.
2023-11-28 | GSE226557 | GEO
Project description:Purpose: Recombinant human erythropoietin administration studies involving “omics” approaches have demonstrated a gene-expression signature that could aid detection of blood doping. However, current anti-doping testing does not involve blood collection into tubes with RNA preservative. This study investigated if whole blood in long-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from thirteen male healthy individuals. Long-term storage: whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., −80°C) storage and RNA extracted. After storage, Tempus and K2EDTA tubes were thawed and extracted using Tempus™ Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA). Samples from seven subjects that presented higher RIN value (≥7) were selected for RNA_Seq analysis. Results: The experiment provided RNA quality and purity for gene expression analysis. Total of 19239 genes were mapped and the gene expression analysis showed that 658 genes were differentially expressed (which means 3.4% of mapped genes). With 269 being up-regulated and 389 down-regulated. None of the transcripts described in previous studies as biomarkers for blood doping (Durussel et al. 2016; Wang, Durussel, et al. 2017) were differently expressed. Conclusion: RNA quantity, purity and integrity was not significantly compromised from long-term storage in blood storage tubes lacking RNA stabilisation, indicating that transcriptomic/omics analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.
2021-09-03 | GSE183073 | GEO
Project description:A total proctocolectomy with ileal pouch-anal anastomosis (IPAA) is considered the surgery of choice for definitive management of familial adenomatous polyposis (FAP) and selected patients with ulcerative colitis (UC). However, this surgical treatment often associates with a long-term complication, pouchitis, which occurs mostly in UC patients. To better define the molecular background of pouchitis, the microarray-based survey was performed using pouch mucosal samples collected from 28 and 8 patients operated for UC and FAP, respectively. A number of 4771 genes was significantly differentiating uninflamed from inflamed mucosal samples, and their functional features were represented mostly by alerted metabolic and cell proliferation pathways. In contrast, functional analyses of aberrantly expressed probe sets between UC and FAP samples, irrespectively of mucosal inflammation status, revealed multiple pathways and terms which were linked to changes in immune response. Noteworthy, the comparison of uninflamed UC and FAP samples distinguished a set of 26 altered mRNAs including inflammation-related transcript encoding a Charcot-Leyden crystal (CLC) protein. The most discrete changes in gene expression profiles differentiating uninflamed UC and FAP mucosal samples were attributed to a Gene Ontology category innate immune response. Our study confirmed alterations of the immune responses as dominant in UC pouchitis which were earlier found in the studies using analyses of singular molecular elements. This observation may be important when managing IPAA patients. Each sample represents three biopsies taken from single patient, from the same areas of the lower part of the pouch mucosa (above the rectal cuff) during endoscopic examination. 28 patients underwent surgery for UC and 8 patients for FAP.
2014-05-01 | E-GEOD-50788 | biostudies-arrayexpress
Project description:Peripheral Blood gene expression is widely used in the discovery of biomarkers and development of therapeutics. Recently, a spate of commercial blood collection and preservation systems have been introduced with proprietary variations that may differentially impact the transcriptomic profiles. Comparative analysis of these collection platforms will help optimize protocols to detect, identify, and reproducibly validate true biological variance among subjects. In the current study, we tested two recently introduced whole blood collection methods, RNAgard® and PAXgene™ RNA, in addition to the traditional method of peripheral blood mononuclear cells (PBMCs) separated from whole blood and preserved in Trizol reagent. Study results revealed striking differences in the transcriptomic profiles from the three different methods that imply ex vivo changes in gene expression occurred during the blood collection, preservation, and mRNA extraction processes. When comparing the ability of the three preservation methods to accurately capture individuals’ expression differences, RNAgard® outperformed PAXgene™ RNA, and both showed significantly better individual separation of transcriptomic profiles than PBMCs. Hence, our study recommends using a single blood collection platform, and strongly cautions against combining methods during the course of a defined study.
2019-09-20 | GSE103889 | GEO
Project description:Purpose: Recombinant human erythropoietin administration studies involving “omics” approaches have demonstrated a gene-expression signature that could aid detection of blood doping. However, current anti-doping testing does not involve blood collection into tubes with RNA preservative. This study investigated if whole blood in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from fourteen male healthy individuals. Short-term storage: whole blood collected into K2EDTA tubes and subjected to short-term (i.e., at 4°C) storage for 6 hours, 12 hours, 24 hours and 48 hours. After storage, blood from K2EDTA tubes were transferred into Tempus™ Blood tubes, and then extracted using Tempus™ Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA). Samples from four subjects of each time point that presented higher RIN value (≥7) were selected for RNA_Seq analysis. Results: The experiment provided RNA quality and purity for gene expression analysis. Considering 6-hours storage as a reference group, the number of differentially expressed genes were 19, 45 and 70 in comparison to 12, 24 and 48-hours, respectively (which means 0.37, 0.88 and 1.37% of mapped genes). Of the 19 differentially expressed genes in the comparison 6 vs. 12-hours, 9 overlapped with the 45 in the comparison 12 vs. 24-hours. Furthermore, 40 of those 45 overlapped with the 70 differentially expressed in the comparison 6 vs. 48-hours. None of the transcripts described in previous studies (Durussel et al. 2016; Wang, Durussel, et al. 2017) were differently expressed. Conclusion: RNA quantity, purity and integrity was not significantly compromised from short-term storage in blood storage tubes lacking RNA stabilisation, indicating that transcriptomic/omics analysis could be conducted using anti-doping samples collected without RNA preservation.
2021-09-03 | GSE183132 | GEO
Project description:Study question: Does storage time impact on transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? Summary answer: For the first time, we demonstrate that the length of cryostorage has no effect on the gene expression profile of human metaphase II oocytes. What is known already: Oocyte cryopreservation is a largely-used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e., women undergoing gonadotoxic therapies) and oocyte donation programs. Although it is known that cryopreservation negatively impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. Study design, size, duration: This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. Participants/materials, setting, methods: Pools of ?10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on 4x44K v2 microarrays (Agilent). Analyses were performed by R v3.1.3 software using the limma package v3.22.7. Main results and the role of chance: Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle process, response to DNA damage stimulus, cellular response to stress and DNA repair, calcium ion binding, malate dehydrogenase activity, mitochondrial membrane respiratory chain. Among the probes significantly up-regulated in cryopreserved oocytes, 2 corresponded to ovary-specific expressed large intergenic noncoding (linc)RNAs. This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed.
2015-09-19 | E-GEOD-69768 | biostudies-arrayexpress