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GSC RT-PCR amplification of 10 cells (SP & CD8 T cells), single SP cell and single-SP-cell equivalent

ABSTRACT: GSM48315-GSM48332: Ten cells from C57Bl/6 male mouse bone marrow (SP or CD8 T cells) were sorted into individual wells of 96-well plates. The mRNA of these cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48333-GSM48344: Single SP cell from C57Bl/6 male mouse bone marrow were sorted into individual wells of 96-well plates. The mRNA of these single cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48345-GSM48349: Forty bone marrow SP cells from C57Bl/6 male mouse bone marrow, Sca-1 positive and Gr-1 negative, gated on the tip of the SP tail, were sorted into 160 microliters of lysis buffer (40 times the amount used for single cells). 4-microliter aliquots (containing the mRNA equivalent to one single cell) were dispensed into individual wells of 96-well plates. The mRNA contained in each aliquot was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. Detection of the microarray hybridization signals was done according to the standard Affymetrix protocol (antibody amplified). Keywords = HSC Keywords = stem cell Keywords = SP Keywords = side population Keywords = CD8 Keywords = lymphocytes Keywords = global single cell RT-PCR Keywords = GSC RT-PCR Keywords: parallel sample

ORGANISM(S): Mus musculus

PROVIDER: GSE2534 | GEO | 2006/01/02

SECONDARY ACCESSION(S): PRJNA92689

REPOSITORIES: GEO

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Transcription profiling and GSC RT-PCR of mouse bone marrow cells SP and CD8 T cells after amplification, single SP cell and single-SP-cell equivalent to examine diversity of single hematopoietic stem cells

Project description:GSM48315-GSM48332: Ten cells from C57Bl/6 male mouse bone marrow (SP or CD8 T cells) were sorted into individual wells of 96-well plates. The mRNA of these cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48333-GSM48344: Single SP cell from C57Bl/6 male mouse bone marrow were sorted into individual wells of 96-well plates. The mRNA of these single cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48345-GSM48349: Forty bone marrow SP cells from C57Bl/6 male mouse bone marrow, Sca-1 positive and Gr-1 negative, gated on the tip of the SP tail, were sorted into 160 microliters of lysis buffer (40 times the amount used for single cells). 4-microliter aliquots (containing the mRNA equivalent to one single cell) were dispensed into individual wells of 96-well plates. The mRNA contained in each aliquot was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. Detection of the microarray hybridization signals was done according to the standard Affymetrix protocol (antibody amplified).

2008-06-12 | E-GEOD-2534 | biostudies-arrayexpress

Project description:WTCCC2 project Schizophrenia (SP) samples

| EGAD00010000262 | EGA

RNA Sequencing Analysis of T Cell Receptor Alpha and Beta Chain in Thymic WT versus CIC-deficient CD4+ SP Non-Treg and Treg Cells.

Project description:Purpose : The goal of this study is to compare the repertoire of T cell receptor (TCR) alpha and beta chain between WT and CIC-deficient thymic CD4+ single positive (SP) T cells, in both non-Treg and Treg populations, to confirm the effect of CIC deficiency on T cell development in the aspect of TCR repertoire. Methods : Treg (CD4+CD8-CD25+GFP+) and non-Treg (CD4+CD8-GFP-) cells from Foxp3-GFP;Cicf/f and Foxp3-GFP;Cicf/f;Vav1-Cre mice were sorted by MoFlo-XDP (Beckman Coulter). Total RNA was extracted using RiboEX (GeneAll) according to the manufacturer's instructions. RNA was then amplified using a commercially available multiplex primer mix covering the TCR alpha and beta chains in two separate PCR reactions. Reverse transcription and subsequent PCR amplification (RT-PCR1) were performed using the Qiagen OneStep RT PCR mix (Qiagen). The cDNA was selected and unused primers were removed by SPRIselect bead selection (Beckman Coulter) followed by a second round of amplification performed with a pair of primers specific for communal sites engineered onto the 5ʹ end of the C- and V- primers used during RT-PCR1. After library preparation, paired-end sequencing was performed using the Illumina Miseq v3 600-cycle Reagent Kit (Illumina). Results : We observed increased frequency of TCRs with long CDR3 length in CIC-deficient non-Treg and Treg cells. We also found significant alterations in the TCR repertoire of TCR beta chain in CIC-deficient Treg cells compared to WT cells, including altered frequency of V and J chain usage. Conclusions : Our study represents the first comparative analysis of TCR repertoire of thymic CD4 SP cells from WT and hematopoietic lineage cell-specific Cic deficient mice. We concluded that CIC deficiency leads to alterations in TCR repertoire of thymic Treg cells.

2021-05-07 | GSE173996 | GEO

EOM and TA Sp cell comparison

Project description:Using Affymetrix GeneChips, we analyzed expression profiles of SP cells from EOM and TA. 348 differentially expressed transcripts defined the EOM-SP transcriptome: 229 upregulated in EOM-SP and 119 in TA-SP. Keywords: Expression Profiling

2008-10-09 | GSE9294 | GEO

A Highly Sensitive and Specific System for Large-scale Gene Expression Profiling

Project description:Gene expression profiling of very few or even single cells is of particular interest in many applications. However, detection of a large number of mRNA sequences from a small number of cells is limited by the sensitivity of available methods. High-throughput multiplex reverse transcription followed by PCR amplification (RT-PCR) has much to offer to these studies due to its inherent sensitivity, efficiency and cost-effectiveness. A multiplex RT-PCR based high-throughput gene profiling system is described in this communication. With this system >1000 different mRNA species can be amplified in a single tube to a detectable amount. By using specially designed PCR primers, the long-standing low specificity problem associated with high-throughput gene expression profiling has been solved. Amplified sequences are then resolved by microarray with probes that only hybridize to sequences amplified from mRNA. The method is so sensitive that mRNA in single cells can be reliably detected. Differentially expressed genes identified with the high-throughput approach in the breast cancer cell line, MCF-7, and its drug resistant variant, MCF-7/AdrR, could be validated by a different method. The approach may greatly facilitate the analysis of combinatorial expression of known genes in any cells in many important applications with a limited amount of RNA. Keywords: drug resistence

2007-06-01 | GSE5920 | GEO

Easy and Accessible Workflow for Label-Free Single-Cell Proteomics

Project description:Single cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often requires custom instrumentation, consumables and reagents for sample processing and separations, which limits accessibility of SCP to a small number of specialized laboratories. Commercial platforms have become available for SCP cell isolation and sample preparation, but the high cost of these platforms and the technical expertise required for their operation place them out of reach of many interested laboratories. Here, we assessed the new HP D100 Single Cell Dispenser for label-free SCP. The low-cost instrument proved highly accurate and reproducible for dispensing reagents in the 200 nL to 2 µL range. We used the HP D100 to isolate and prepare single cells for SCP within 384-well PCR plates. When the well plates were immediately centrifuged following cell dispensing and again after reagent dispensing, we found that ~97% of wells that were identified in the instrument software as containing a single cell indeed provided proteome coverage expected of a single cell. The D100 Single Cell Dispenser combined with one-step sample processing provides a very rapid easy-to-use workflow for SCP with no reduction in proteome coverage relative to a nanowell-based workflow. The commercial well plates also facilitate autosampling with commercial instrumentation. Single cell samples were analyzed using home-packed 30-µm-i.d. nanoLC columns as well as commercially available 50-µm-i.d. columns. The commercial columns led to identification of ~35% fewer identified proteins. However, combined with the well plate-based preparation platform the presented workflow provides fully commercial and relatively low cost alternative for SCP sample preparations and separations, which should greatly broaden accessibility of SCP to other laboratories.

2023-10-09 | PXD043355 | Pride

Mesenchymal stem cell-derived exosomes stimulate cycling quiescence and early breast cancer dormancy in bone marrow

Project description:Transwell cultures were established in 6-well plates with 0.4 micron inserts. Equal amounts (6x10e4) of BCCs and MSCs were added to the outer and inner wells, respectively, in DMEM with 10% exosome-free FCS. After 24 h, the inner wells with BCCs were discarded. The MSCs were washed with PBS and then incubated with DMEM with 2% exosome-free FCS. At 48 h, exosomes were isolated from the MSC media. The exosomes from triplicate wells were added to duplicate naïve BCCs at 2.5x105/well of 6-well plates.

2016-08-08 | GSE85341 | GEO

Evaluation of SINGLE PORT (SP) Robotic Technology in Colorectal Surgery

Project description:Currently a multiport robotic surgery platform (Intuitive Xi) is widely available and used for colorectal surgery indications. A Single port platform (Intuitive SP) is FDA approved for Head and Neck and Urology but has not been widely used in colorectal surgery. This study seeks to evaluate the safe and effective use of the SP platform for colorectal surgery indications.

| 2718159 | ecrin-mdr-crc

Arabidopsis thaliana small RNAs sequences identified using high-throughput 454 sequencing technology

Project description:To determine the extent to which the major small RNA pathways functions across the Arabidopsis thaliana genome, small RNA populations from several tissues of wild-type (wt) and mutant plants were amplified by RT-PCR and sequenced using high-throughput 454 sequencing technology. Keywords: small RNAs, high-throughput sequencing

2007-02-01 | GSE6682 | GEO

Rhodococcus sp. RHA1 grown on propane versus pyruvate

Project description:three late exponential phase Rhodococcus sp. RHA1 propane-grown cultures compared to pyruvate-grown cultures Keywords: growth substrate

2007-11-01 | GSE8480 | GEO

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