Real-time quantitative PCR analysis of human leukemic B-cells [CC treatment]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE55686 | GEO | 2015/03/07
SECONDARY ACCESSION(S): PRJNA240553
REPOSITORIES: GEO
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Project description:Fresh blood samples were collected from 10 CLL (chronic lymphocytic leukemia) previously untreated patients before and after 2 weeks of the first cycle of CCR (cladribine, cyclophosphamide, rituximab) treatment. Peripheral blood mononuclear cells (PBMNCs) were separated from EDTA blood by layering on the Histopaque 1077 and centrifuging on a density gradient. Mean B-cell CD19+ purity was ≥95% as measured by flow cytometry (FC). Microarray analysis was performed using 0.5 microgram of RNA transcribed to cDNA. Prepared cDNA samples were subjected to RT-PCR in duplicate in the TaqMan 7900HT Sequence Detection System (Applied Biosystems).
2013-12-31 | GSE33925 | GEO
Project description:Fresh blood samples were collected from 8 CLL (chronic lymphocytic leukemia) previously untreated patients before and after 2 weeks of the first cycle of CC (cladribine, cyclophosphamide) treatment. Peripheral blood mononuclear cells (PBMNCs) were separated from EDTA blood by layering on the Histopaque 1077 and centrifuging on a density gradient. Mean B-cell CD19+ purity was ?95% as measured by flow cytometry (FC). Microarray analysis was performed using 0.5 microgram of RNA transcribed to cDNA. Prepared cDNA samples were subjected to RT-PCR in duplicate in the TaqMan 7900HT Sequence Detection System (Applied Biosystems). qPCR gene expression profiling. Intensity of gene expression in CLL cells collected from particular patient before treatment was compared with the gene expression signals in CLL cells from the same patient after 2 weeks of treatment.
2015-03-07 | E-GEOD-55686 | biostudies-arrayexpress
Project description:Fresh blood samples were collected from 10 CLL (chronic lymphocytic leukemia) previously untreated patients before and after 2 weeks of the first cycle of CCR (cladribine, cyclophosphamide, rituximab) treatment. Peripheral blood mononuclear cells (PBMNCs) were separated from EDTA blood by layering on the Histopaque 1077 and centrifuging on a density gradient. Mean B-cell CD19+ purity was ≥95% as measured by flow cytometry (FC). Microarray analysis was performed using 0.5 microgram of RNA transcribed to cDNA. Prepared cDNA samples were subjected to RT-PCR in duplicate in the TaqMan 7900HT Sequence Detection System (Applied Biosystems). qPCR gene expression profiling. Intensity of gene expression in CLL cells collected from particular patient before treatment was compared with the gene expression signals in CLL cells from the same patient after 2 weeks of treatment.
2013-12-31 | E-GEOD-33925 | biostudies-arrayexpress
Project description:Human Peripheral blood mononuclear cells (PBMC) were isolated from healthy human whole blood by density gradient centrifugation using Histopaque®-1077 and cultured in RPMI (containing 10% FBS) with 10 ng/ml human recombinant GM-CSF at 37 °C for 7 days. These cells are confirmed as human macrophages. These macrophages were transfected with vector control, miR-466l,anti-miR-negative or anti-miR-466l (37 °C, 72h). After transfection, total RNA was extracted for reverse transcription. The product cDNA was collected for real-time PCR with respective primers.
2013-11-08 | GSE52174 | GEO
Project description:Human Peripheral blood mononuclear cells (PBMC) were isolated from healthy human whole blood by density gradient centrifugation using Histopaque®-1077 and cultured in RPMI (containing 10% FBS) with 10 ng/ml human recombinant GM-CSF at 37 °C for 7 days. These cells are confirmed as human macrophages. These macrophages were transfected with vector control, miR-466l,anti-miR-negative or anti-miR-466l (37 °C, 72h). After transfection, total RNA was extracted for reverse transcription. The product cDNA was collected for real-time PCR with respective primers. In the study presented here, lentiviral vector control (VC), miR-466l, anti-miR-negative (anti-miR-neg) or anti-miR-466l transfected human macrophages were used to acquire expression profiles of a total of 60 unique genes related to inflammation
2013-11-08 | E-GEOD-52174 | biostudies-arrayexpress
Project description:The human lung cancer cell line A549 was cultured in media alone, with cyclophosphamide (CPM), oxaliplatin (OXP) or DMSO alone for 72 hours. The exhausted culture media were aspirated and stored at -20C. Peripheral blood mononuclear cells (PBMCs) were isolated from four pathologically healthy donor buffy coats using Histopaque-1077. Following removal of red blood cell and platelet contamination, monocytes were isolated using magnetic beads coated with anti-CD14. Cells were resuspended and cultured in a DC-maturing medium for 7 days. Non- and loosely-adherent cells (the DC fraction) were harvested and purity assessed by CD11c/HLA-DR/CD14 immuno-discrimination by flow cytometry. 1x10^5 unstimulated DCs were cultured with tumour derived supernatants or culture media alone for 24 hours. DCs were then harvested and RNA isolated by Trizol. Total RNA was labelled and hybridised to Illumina Human HT-12v3 arrays. A separate aliquot of DCs from each patient was exposed to supernatant derived from tumour treated with OXP; changes in CD80, CD83 and CD86 were measured and DC populations showing a greater than two-fold up-regulation of these markers were classified as "GOOD_DC".
2011-04-01 | E-MTAB-389 | biostudies-arrayexpress
Project description:Purpose: Circulating hematopoietic stem/progenitor cells (cHSPCs) are rare in normal human peripheral blood (PB) without mobilization. We apply a novel three-dimension culture system (3DCS) seeded with no mobilized PB monocytes (PBMNCs), to expand cHSPCs. Two-dimension culture system (2DCS), primary PBMNCs and CD34+ HSPCs derived from bone marrow serves as system, negative and positive control groups respectively. Bulk RNA sequencing analysis is performed to dissect the function and molecular mechanism for cHSPCs. Methods: Four groups are designed in the study including the samples cultured in 2DCS, cultured in 3DCS, normal primary PBMNCs and CD34+ HSPCs derived from mobilized peripheral blood. Total RNA was extracted from the cells followed by mRNA enrichment with poly-T oligo-attached magnetic beads. Cells from 2DCS and normal primary PBMNCs served as controls (the number of repetitions for the groups, n = 3). Mobilized bone marrow-derived CD34+ HSPCs were purified with EasySep Human CD34 Positive Selection Kit (StemCell Technologies) serving as positive control (n = 4). Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit (NEB, Illumina®, USA) following manufacturer’s recommendations. Index codes were added to attribute sequences to each sample. Preferential 150~200 bp in length of cDNA fragments were selected and purified with AMPure XP system (Beckman Coulter, Beverly, USA). PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and index (X) Primer after adaptor of cDNA being ligated with USER Enzyme (NEB, USA). At last, PCR products were purified with AMPure XP system and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded products was carried out on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. Sequencing raw data were generated using the Illumina HiSeqTM platform, and 125 bp/150 bp paired-end reads were selected. Results: Bulk RNA sequencing analysis reveals that the samples in 3DCS have more similarity expression pattern with CD34+ hematopoietic stem/progenitor cells (HSPCs) than ones in 2DCS and PBMNCs, for their expression higher level of the specific hematopoietic stem signatures including core stem transcription factors (TFs) as Runx1, HoxA9, HoxB4 and GATA2, self-renewal genes, surface epitopes, and some signaling pathways essential for HSPC fate determination. This study provides a novel culture system to expand rare cHSPC in normal PB, and the underlying molecular and functional signatures are dissected. Conclusions: Our study demonstrates the first detailed analysis of normal peripheral blood-derived cHSPC transcriptomes based on RNA-seq technology. Our results show that 3DCS facilitates to expand the rare circulating HSPCs with repopulating capacity. The underlying molecular and functional signatures of cHSPCs are dissected. The study provides a platform for normal PB further clinical applications in cell transplantation and personalized precision medicine.
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Project description:Early EPCs (eEPCs) appear at less than 1 week in culture dishes, whereas late EPCs (LEPCs) appear late at 2-4 weeks. Distinct angiogenic properties between these two EPC subpopulations have been disclosed by the angiogenesis assay: late EPCs, but not eEPCs, form vascular networks de novo and are able to incorporate into vascular networks. On the contrary, eEPCs, but not late ones, indirectly augment tubulogenesis even when physically separated by a Transwell membrane, implying the involvement of a cytokine-based paracrine mechanism. Microarrays are great tools to M-bM-^@M-^\snapshotM-bM-^@M-^] the transcriptome profiles, and thus provide deeper insights into gene expressions and microRNA-gene interactions. Cord blood mononuclear cells (MNCs) were Histopaque-1077 isolated and plated in endothelial growth medium-2 on fibronectin-coated six-well plates at 37M-BM-0C. After 3 days of culturing, attached eEPCs appeared, and medium and nonadherent cells were removed. Thereafter, the medium were replaced every 2 days, and a certain number of eEPCs continued to grow into the LEPCs colonies, which emerge 2M-bM-^@M-^S4 weeks after the start of MNC culture. Total RNA of early and late EPCs were extracted and evaluated with Affymetrix GeneChip Human U133 plus 2.0
2014-04-03 | E-GEOD-37045 | biostudies-arrayexpress
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2015-01-26 | E-GEOD-59635 | biostudies-arrayexpress
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2015-01-26 | E-GEOD-59654 | biostudies-arrayexpress