Background This report describes a method for the generation of global

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. step in the workflow was validated and the sorting/storage conditions optimized as described in this report. 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). 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?BML-190 in normal bone marrow and tonsil samples eight evaluated genes were expressed in accordance with the biology of lymphopoiesis (p-values?DNM1 omits an immunomagnetic purification step for B-cell enrichment before FACS as often performed [17-20] which is problematic when BML-190 low frequent B-cells are sorted. Methods Protocol overview A flow chart of the established protocol and methods for sorting the B-cell subsets in different tissue [see Additional file 1]. Impact of amplification Gene specific amplification determined by qPCRTotal RNA from one million cells was extracted from BML-190 six multiple myeloma (MM) cancer cell lines (CCLs) (MOLP-8 KMS-12-BM RPMI-8226 OPM-2 LP-1 and KMM-1) using RNAeasy Plus Micro equipment (QIAGEN Hilden Germany). Genomic DNA was removed using gDNA Eliminator Spin Columns (QIAGEN Hilden Germany). RNA quality was evaluated with an Agilent 2100 bioanalyzer (Agilent Technologies Inc. Palo Alto CA) (RIN?>?9.8). Total RNA from each CCL was processed in parallel by either directly converting 500?ng to cDNA (non-amplified) with SuperScript III First-Strand Synthesis Supermix (Invitrogen Paisley UK) or by amplifying 5?ng with an Ovation Pico WTA system (NuGEN Technologies Inc. San Carlos CA) as described by the manufacturer. QPCR assays were performed by BML-190 comparing amplified cDNA at 25?ng/reaction to non-amplified cDNA derived from SuperScript III at 25?ng/reaction (total RNA equivalents). Commercially available Taqman primer probes sets previously described in the qPCR Section were used. Comparing NuGEN protocol to standard protocolTotal RNA from one million cells of the same four CCLs KMM-1 OPM-2 U2932_M and SU-DHL-5 was subjected to the NuGEN protocol or to the standard protocol from Ambion (Ambion WT Expression kit Ambion Inc. Austin TX) following the manufactures recommendations. The input of total RNA was 1?ng for the NuGEN protocol whereas the input for the Ambion protocol was 100?ng. Optimisation of storage buffer Selection of storage buffer for sorted cellsThe storage buffer was examined by sorting 15 0 fresh naive tonsil cells from a single donor directly into 12 separate tubes containing 450?μl of either lysis/binding buffer RNAlater (Ambion Austin TX) or PrepProtect. mRNA was isolated using the μMACS? technology (Miltenyi Biotech Bergisch-Gladbach Germany) allowing isolation on μ Columns and elution with pure water. This technology is referred to as magnetic bead.