Validation and implementation of a method for microarray gene expression profiling of minor B-cell subpopulations in man

This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to o...

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Bibliographic Details
Published in:BMC immunology 2014-01, Vol.15 (1), p.3-3, Article 3
Main Authors: Bergkvist, Kim Steve, Nyegaard, Mette, Bøgsted, Martin, Schmitz, Alexander, Bødker, Julie Støve, Rasmussen, Simon Mylius, Perez-Andres, Martin, Falgreen, Steffen, Bilgrau, Anders Ellern, Kjeldsen, Malene Krag, Gaihede, Michael, Nørgaard, Martin Agge, Bæch, John, Grønholdt, Marie-Louise, Jensen, Frank Svendsen, Johansen, Preben, Dybkær, Karen, Johnsen, Hans Erik
Format: Article
Language:English
Subjects:
Analysis
Antigens, CD - metabolism
B-Lymphocyte Subsets - metabolism
Biological products industry
Bone marrow
Cancer
Flow Cytometry
Gene expression
Gene Expression Profiling - methods
Genetic aspects
Hematology
Humans
Lymphoid Tissue - cytology
Lymphoid Tissue - metabolism
Medical equipment and supplies industry
Medical research
Medical test kit industry
Messenger RNA
Methods
Oligonucleotide Array Sequence Analysis
Organ Specificity - genetics
Principal components analysis
Real-Time Polymerase Chain Reaction
Reproducibility of Results
Statistical analysis
Surgery
Transcription factors
Variance analysis
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Summary:This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to optimize and validate each step in the procedure. Normal lymphoid tissues from blood, tonsils, thymus and bone marrow were immunophenotyped by the 8-colour Euroflow panel using multiparametric flow cytometry. Subsets of B-cells containing cell numbers ranging from 800 to 33,000 and with frequencies varying between 0.1 and 10 percent were sorted, subjected to mRNA purification, amplified by the NuGEN protocol and finally analysed by the Affymetrix platform. Following a step by step strategy, each step in the workflow was validated and the sorting/storage conditions optimized as described in this report. First, an analysis of four cancer cell lines on Affymetrix arrays, using either 100 ng RNA labelled with the Ambion standard protocol or 1 ng RNA amplified and labelled by the NuGEN protocol, revealed a significant correlation of gene expressions (r ≥ 0.9 for all). Comparison of qPCR data in samples with or without amplification for 8 genes showed that a relative difference between six cell lines was preserved (r ≥ 0.9). Second, a comparison of cells sorted into PrepProtect, RNAlater or directly into lysis/binding buffer showed a higher yield of purified mRNA following storage in lysis/binding buffer (p < 0.001). Third, the identity of the B-cell subsets validated by the cluster of differentiation (CD) membrane profile was highly concordant with the transcriptional gene expression (p-values
ISSN:1471-2172
1471-2172
DOI:10.1186/1471-2172-15-3