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


METHODS
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.


RESULTS
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 <0.001). Finally, in normal bone marrow and tonsil samples, eight evaluated genes were expressed in accordance with the biology of lymphopoiesis (p-values < 0.001), which enabled the generation of a gene-specific B-cell atlas.


CONCLUSIONS
A description of the implementation and validation of commercially available kits in the laboratory has been examined. This included steps for cell sorting, cell lysis/stabilization, RNA isolation, RNA concentration and amplification for microarray analysis. The workflow described in this report will enable the generation of microarray data from minor sorted B-cell subsets.
Kim Steve
KS
Bergkvist
Mette
M
Nyegaard
Martin
M
Bøgsted
Alexander
A
Schmitz
Julie Støve
JS
Bødker
Simon Mylius
SM
Rasmussen
Martin
M
Perez-Andres
Steffen
S
Falgreen
Anders Ellern
AE
Bilgrau
Malene Krag
MK
Kjeldsen
Michael
M
Gaihede
Martin Agge
MA
Nørgaard
John
J
Bæch
Marie-Louise
ML
Grønholdt
Frank Svendsen
FS
Jensen
Preben
P
Johansen
Karen
K
Dybkær
Department of Haematology, Aalborg University Hospital Science and Innovation Center, Sdr Skovvej 15, DK-9000 Aalborg, Denmark. haej@rn.dk.
Hans Erik
HE
Johnsen
D015703:Antigens, CD / Q000378:metabolism; D016175:B-Lymphocyte Subsets / Q000378:metabolism*; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008221:Lymphoid Tissue / Q000166:cytology / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D009928:Organ Specificity / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
D015703:Antigens, CD