AbstractRecovery of the blood and immune system after chemotherapy requires proliferation of hematopoietic stem and progenitor cells (HPSCs). It has been shown that systemically released factors in serum after chemotherapy stimulate HSPC expansion in vitro. We wondered if microRNAs (miRNAs) circulating in serum could account for this effect. Therefore, we compared the miRNA expression profiles of serum from patients with hematologic malignancies before and after chemotherapy. In addition to a general decrease in miRNA expression after chemotherapy, we found 23 miRNAs to be significantly differentially expressed in serum before versus after chemotherapy. The miRNA microarray data are available at NCBI's Gene Expression Omnibus (GEO) Series accession number GSE57570. Here, we provide a detailed protocol of the miRNA microarray and data analysis

Bissels, Ute

Bosio, Andreas

Brümmendorf, Tim Henrik

Diener, Yvonne

Jost, Edgar

Wagner, Wolfgang

Walenda, Thomas

Genomics Data

PubMed

Publikationsserver der RWTH Aachen University

MicroRNA expression profiles of serum from patients before and after chemotherapy

Brümmendorf, Tim H.

Elsevier - Publisher Connector 

Genomics Data 6 (2015) 125–127
Contents lists available at ScienceDirect
Genomics Data
j ou rna l homepage: ht tp : / /www. journa ls .e lsev ie r .com/genomics-data /Data in BriefMicroRNA expression proﬁles of serum from patients before and
after chemotherapyYvonne Diener a,⁎, Thomas Walenda b, Edgar Jost c, Tim H. Brümmendorf c, Andreas Bosio a,
Wolfgang Wagner b, Ute Bissels a
a Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
b Helmholtz Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany
c Department for Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, RWTH Aachen University Medical School, Aachen, GermanySe
Se
D
E
E
C
Sa
⁎ Corresponding author.
http://dx.doi.org/10.1016/j.gdata.2015.08.018
2213-5960/© 2015 The Authors. Published by Elsevier Inca b s t r a c ta r t i c l e i n f oArticle history:
Received 14 August 2015
Accepted 15 August 2015
Available online 2 September 2015
Keywords:
MicroRNA
Hematopoietic stem cells
Serum
MicroarrayRecovery of the blood and immune system after chemotherapy requires proliferation of hematopoietic stem and
progenitor cells (HPSCs). It has been shown that systemically released factors in serum after chemotherapy stim-
ulate HSPC expansion in vitro. Wewondered if microRNAs (miRNAs) circulating in serum could account for this
effect. Therefore, we compared the miRNA expression proﬁles of serum from patients with hematologic malig-
nancies before and after chemotherapy. In addition to a general decrease inmiRNA expression after chemother-
apy, we found 23miRNAs to be signiﬁcantly differentially expressed in serum before versus after chemotherapy.
The miRNA microarray data are available at NCBI's Gene Expression Omnibus (GEO) Series accession number
GSE57570. Here, we provide a detailed protocol of the miRNA microarray and data analysis.
© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).SpeciﬁcationsOrganism/cell
line/tissueHomo sapiens (serum)x Male and female
quencer or
array typeAgilent Human miRNA Microarray Release 16.0, 8 × 60 Kata format Raw and processed
xperimental
factorsSerum before vs. after chemotherapyxperimental
featuresProﬁling of circulating microRNAs in serum from patients with
different hematologic malignancies before and after
chemotherapy as well as healthy donorsonsent Allowed for reuse citing original authors
mple source
locationAachen, Germany1. Direct link to deposited data
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57570
2. Experimental design, materials and methods
2.1. Experimental design
Chemotherapy and hematopoietic stem cell transplantation (HSCT)
are standard therapies for the treatment of hematologic malignancies.. This is an open access article underThe subsequent recovery of the blood and immune system requires
activation and proliferation of hematopoietic stem and progenitor
cells (HSPCs). Walenda and colleagues [1] demonstrated that systemi-
cally released factors in serum from patients after chemotherapy stimu-
late in vitro expansion of HSPCs. MicroRNAs (miRNAs) have been found
to circulate in serum and their expression levels vary in physiological
and pathological conditions [2,3]. Furthermore, they are important
regulators of cell fate and are able to inﬂuence hematopoietic stem
cell proliferation and differentiation [4]. In this study [5], we have com-
pared the miRNA expression proﬁles of serum from nine patients with
lymphoma, acute myeloid leukemia (AML) or multiple myeloma
(MM) before and after therapy as well as seven healthy donors as
control. In summary, the presence of 1205 human and 144 human
viral miRNAs was analyzed using the Agilent Human microRNA Micro-
array platform (Rel. 16).
2.2. Sample preparation
Serum samples from seven healthy donors and nine patients with
different hematologic malignancies were collected at different time
points in the course of therapy after informed written consent as
described before [1]. Ten milliliters of peripheral blood was collected
before chemotherapy and during leukopenia after chemotherapy and
transferred into a 15 mL tube (Greiner). For preparation of serum, the
blood was agitated horizontally at 37 °C for 1 h to allow coagulation
and incubated upright at 4 °C for 4 h. After centrifugation for 15 min
at 840 ×g, the serum supernatant was carefully removed, aliquotedthe CC BY license (http://creativecommons.org/licenses/by/4.0/).
126 Y. Diener et al. / Genomics Data 6 (2015) 125–127and stored at−80 °C until use. Further information on serum samples
and patients was published by Walenda et al. [5]. For total RNA extrac-
tion, 500 μL of frozen serum was thawed for 30 min at room tempera-
ture and mixed with three volumes of TRIzol LS Reagent (Ambion).
RNA was isolated using the miRNeasy mini kit (Qiagen) according to
the manufacturer's instructions. The volume of the obtained RNA
solution was reduced in a Speed Vac vacuum concentrator to a ﬁnal
volume of 1 μL.
2.3. RNA labeling and microRNA microarray hybridization
The Human microRNA Microarray Kit (Rel16.0, Agilent Technolo-
gies) was used for labeling and hybridization according to the
manufacturer's protocol. In brief, 1 μL of total RNA was labeled with Cy-
anine 3 (Cy3), resuspended in hybridization buffer and hybridized to
the array platform overnight (20 hours) at 55 °C in a rotating Agilent
hybridization oven using Agilent's recommended hybridization cham-
ber. Subsequently, the microarrays were washed with the Agilent
Gene Expression Wash Buffer 1 for 5 min at room temperature. A sec-
ond washing step was performed with Agilent Gene Expression Wash
Buffer 2warmed to 37 °C for 5min. Fluorescence signals after hybridiza-
tion were detected with a DNA microarray scanner G2505C (Agilent
Technologies) using one color scan setting for 8 × 60 K array slides
(Scan Area 61 × 21.6 mm, Scan resolution 3 μm, Dye channel is set to
Green and Green PMT is set to 100%).
2.4. Microarray data analysis
In order to obtain background subtracted and outlier rejected
signal intensities, the scanned microarray images were analyzed and
processed with the Agilent feature extraction software (v10.7.3.1)
using default parameters (protocol miRNA_107_Sep09 and Grid:
031181_D_F_20111226). The resulting rawTotal Gene Signal intensities
(SI, gTotalGeneSignal column) were exported to Microsoft Excel and
ﬁltered for detected miRNAs (SI ≠ 0.1). Analysis of the detected miRNAsFig. 1. Higher number of detected microRNAs in patients' sera before chemotherapy.
VENN diagram presenting numbers of detected miRNAs in serum from healthy donors
(healthy), patients before (BC) and after chemotherapy (AC). Total gene intensities were
ﬁltered for miRNAs with median expression ≠0.1 over all donors. Exclusively in healthy
donors detected miRNAs (8) were either detected with low signal intensities (b10) or
characterized as viral RNAs (HSV and KSHV).revealed a lower number ofmiRNAs in serum taken after chemotherapy
compared to serum before chemotherapy (Fig. 1) as well as decreased
miRNA signal intensities after chemotherapy (Fig. 2A). This global
decrease inmiRNAexpression levels signiﬁcantly correlatedwith leuco-
cyte numbers in the patients' blood, which might indicate that the
observed alterations are caused by leukopenia. As we were interested
in miRNAs signiﬁcantly differentially expressed in serum before versus
after chemotherapy independently from the disturbance in blood cell
counts, the data were normalized to the array median as follows: (1) a
ﬁlter was set for miRNAs which were detected with an SI ≠ 0.1 in all
patient samples before and after chemotherapy (= 18 samples),
giving 66 miRNAs in total (including Agilent positive controls: dmr,
hur). (2) For each sample, the median over these 66 miRNAs was
calculated. (3) The value of each single miRNA was divided through
the second highest median value. The highest median value was not
used as it presumably resulted from an outlier. For statistical analysis
and to determine differentially expressed miRNAs, median normalized
signal intensities were log2 transformed and subjected to Student's
paired t-test usingMicrosoft Excel 2010.We found 23miRNAs to be sig-
niﬁcantly differentially expressed in serum before versus after chemo-
therapy (P ≤ 0.01, Fig. 2B). The three most signiﬁcantly differentially
expressed miRNAs were miRNA-320c (P = 0.0002), miRNA-1275
(P = 0.0005), and miRNA-3663-3p (P = 0.0006), among which miR-
320c and miR-1275 were downregulated and miR-3663-3p was upreg-
ulated after chemotherapy.
The hybridization protocol, raw and normalized data are provided in
NCBI's Gene Expression Omnibus (GEO, Series accession number
GSE57570).
3. Discussion
Our data show that miRNA expression proﬁles in serum change in
the course of treatment of hematologic diseases. Overall miRNA levels
in patient sera were elevated prior to chemotherapy and compared to
healthy controls, in accordance with aberrant miRNA expression and
disease-speciﬁc miRNAs which have been described in various hemato-
logic disorders such as AML,MM [6] and ALL [7]. However, we observed
that the number of detectedmiRNAs correlatedwith the patients' blood
cell count. This is in line with previously described ﬁndings, that the
majority of circulating miRNAs originates from blood cells and that
disturbance in blood cell counts can inﬂuence miRNA expression in
serum [8]. Taking this into account, we normalized the expression pro-
ﬁles in order to identify speciﬁc miRNAs that signiﬁcantly changed
before versus after therapy. Normalization of miRNA data from serum
samples is not trivial as standard normalization methods are not appli-
cable here. Quantile normalizationwhich is commonly used for process-
ing of gene expression data is critical because miRNA expression data
sets contain comparatively few data points [9]. Normalization using
small nuclear RNAs such as RNU6B is not recommended as their expres-
sionwas found to be dysregulated under pathological conditions [10] or
they are not detectable in serum [11]. Another common method is the
normalization to endogenous reference miRNAs such as miRNA-16,
but it has been shown that the expression of this reference miRNA can
be unstable or inﬂuenced by hemolysis [8,12]. Thus, we decided to
normalize our data to the array median prior to identiﬁcation of
miRNA candidates. Subsequent statistical analysis revealed 23 signiﬁ-
cantly differentially expressed miRNAs, of which miRNA-320c showed
the highest signiﬁcance and was chosen for further analysis such as
qRT-PCR, target prediction and biological function in HSPCs [5].
Conﬂict of interest
The authors declare that Yvonne Diener, Ute Bissels and Andreas
Bosio are employed at Miltenyi Biotec GmbH. Wolfgang Wagner is
involved in the Cygenia GmbH. No further competing interests exist.
Fig. 2.Median normalization of miRNA microarray data enabled identiﬁcation of signiﬁcantly differentially expressed miRNAs. Median signal intensities of miRNAs in serum after (AC)
versus before chemotherapy (BC, n = 9), before (A) and after median normalization (B). White dots represent signiﬁcantly differentially expressed microRNAs after normalization.
Data points on the x-axis represent 163 miRNAs that are exclusively detected in BC serum.
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MicroRNA expression profiles of serum from patients before and after chemotherapy 

Recovery of the blood and immune system after chemotherapy requires proliferation of hematopoietic stem and progenitor cells (HPSCs). It has been shown that systemically released factors in serum after chemotherapy stimulate HSPC expansion in vitro. We wondered if microRNAs (miRNAs) circulating in serum could account for this effect. Therefore, we compared the miRNA expression profiles of serum from patients with hematologic malignancies before and after chemotherapy. In addition to a general decrease in miRNA expression after chemotherapy, we found 23 miRNAs to be significantly differentially expressed in serum before versus after chemotherapy. The miRNA microarray data are available at NCBI's Gene Expression Omnibus (GEO) Series accession number GSE57570. Here, we provide a detailed protocol of the miRNA microarray and data analysis

Yvonne Diener

Thomas Walenda

Edgar Jost

Tim H. Brümmendorf

Andreas Bosio

Wolfgang Wagner

Ute Bissels

Directory of Open Access Journals

RWTH Publications

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MicroRNA expression profiles of serum from patients before and after chemotherapy

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