Effects of magnetic bead-based enrichment of NB cells on their gene expression.

<p>qPCR arrays were used to analyze the effects of magnetic bead-based enrichment on the expression of 71 genes in NB cells. In (a) the altered gene expression is shown for cells that have been enriched only once after density gradient separation, whereas in (b) the effect of two following magnetic bead-based enrichment steps is shown. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given conditions compared to the baseline (LAN-1 cells before spiking into PB). The expression of genes with |log₂FC|>1 but p>0.05 are not considered as significant, as their expression was not coherently changed in the different biological replicates. The log₂ fold change is indicated on the y-axis and the mean Ct values in the x-axis.</p

Effects of magnetic bead-based enrichment of NB cells on their gene expression.

<p>qPCR arrays were used to analyze the effects of magnetic bead-based enrichment on the expression of 71 genes in NB cells. In (a) the altered gene expression is shown for cells that have been enriched only once after density gradient separation, whereas in (b) the effect of two following magnetic bead-based enrichment steps is shown. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given conditions compared to the baseline (LAN-1 cells before spiking into PB). The expression of genes with |log₂FC|>1 but p>0.05 are not considered as significant, as their expression was not coherently changed in the different biological replicates. The log₂ fold change is indicated on the y-axis and the mean Ct values in the x-axis.</p

Altered gene expression of NB cells kept at 4°C and at RT up to 72h.

<p>70 genes were analyzed by qPCR array. The altered gene expression of LAN-1 cells kept for 24 hours in PB on 4°C (a) and at room temperature (b) is shown. In (c) and (d) the same is shown for 48h, and in (e) and (f) for 72h. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given time points compared to the baseline (time point 0h). Genes with |log₂FC|>1 and labeled by black dots, did not show significant changes (p>0.05) between the three biological replicates. The log₂ fold change for a given conditions is indicated on the y-axis, whereas the mean Ct value is shown on the x-axis.</p

Experimental design.

<p><b>(a)</b> We spiked LAN-1 NB cells into fresh PB and kept the samples for 0, 24, 48 and 72h at room temperature and, for the same time periods, at 4°C prior to density gradient separation. The LAN-1 cells were enriched from the MNC fraction with magnetic beads to a 99% purity of the tumor cell fractions prior to homogenization in TRIzol. RNA was isolated from all seven samples simultaneously and used for the qPCR array. (<b>b)</b> LAN-1 cells were spiked into PB and tumor-free BM, and density gradient separation was immediately performed. The MNCs were frozen in 20% DMSO for seven days at -80°C. After thawing, the LAN-1 cells were either directly enriched by magnetic bead-based separation, or an additional density gradient separation (*) was performed prior to magnetic bead-based separation. The samples were homogenized in TRIzol and the isolated RNA was used for qPCR (in case of PB) and microarrays (in case of BM). <b>(c)</b> LAN-1 cells were spiked into PB and density gradient separation of MNCs was performed without delay, following two enrichment steps in a row. The >99% LAN-1 cell fractions were homogenized in TRIzol and RNA was isolated from all samples simultaneously. qPCR arrays were performed in order to analyze the effect of enrichment on selected genes.</p

Unsupervised clustering of NB samples after freezing, storage and thawing procedure.

<p>Microarray analysis of three biological replicates (A-C) and the three different pretreatment conditions: fresh (no freezing/thawing), frozen 1 (thawing > magnetic bead-based separation of LAN-1 cells) and frozen 2 (thawing > density gradient separation > magnetic bead-based separation of LAN-1 cells). In the unsupervised clustering of the expression of the analyzed genes, the fresh and the two differently frozen samples (1, 2) did not cluster. The correlation coefficient (R) is illustrated by the color key: white (0) = no correlation and red (1) = high correlation.</p

Effects of freezing, storage and thawing of samples on NB gene expression.

<p>In (a-b) we analyzed the effect of freezing, storage at -80°C and thawing by qPCR array on the expression of 64 genes of NB cells spiked into PB. In (a) we show the effect when after thawing the samples were only enriched by magnetic bead-based separation, whereas in (b) we introduced an additional density gradient separation prior to magnetic bead-based separation. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given conditions compared to the baseline (LAN-1 cells enriched and homogenized in TRIzol immediately after spiking). Genes with |log₂FC|>1 and p>0.05 are represented by black dots (not significant), as their expression was not coherently changed between the three biological replicates. The log₂ fold change is indicated on the y-axis and the mean Ct values in the x-axis. In (c-d) we analyzed the same effects on NB cells spiked into BM by microarrays. The mean log₂ expression is shown in the x-axis, and the log₂ fold change on the y-axis.</p