Supplementary text
Methods
Isolation of mantle B cells
Cell suspensions were obtained by mincing and filtering the tissue in ice-cold culture medium
(RPMI). Mononuclear cells were isolated by Ficoll-Paque Plus (Amersham Pharmacia Biotech
AB, Uppsala, Sweden). Tonsil single-cell suspensions were incubated with CD19 (PE-Cy™7
Mouse Anti-Human CD19, BD Franklin Lakes, NJ, USA), IgD (FITC Mouse Anti-Human IgD, BD)
and CD27 (APC Mouse anti-Human CD27, BD) antibodies for 25 min at room temperature.
CD19+/IgD+/CD27- cells were sorted, yielding a purity of at least 95%.
RNA extraction
For miRNA hybridization and quantitative PCR of fresh frozen samples, total RNA was isolated
by Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality was checked using total RNA
(small fraction chip) with the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara,
CA, USA) and by 1% agarose electrophoresis, following the standard procedure.
For gene expression studies, RNA extracted with Trizol was purified by RNase-free DNase I
RNeasy kit (Qiagen Inc., Valencia, CA, USA) following the standard procedure. mRNA quality
was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies).
For quantitative PCR of FFPE samples RNA was extracted as previously described (1, 2) with
minor modifications. In brief, three 10μm tissue sections were deparaffinized by xylene
incubation. After centrifugation and an ethanol washing and drying procedure, the pellet was
resuspended in the lysis buffer and incubated with proteinase K (Quiagen). RNA was purified
by phenol-chloroform extraction followed by isopropanol precipitation.
1
Microarray procedures: mRNA hybridization
RNA for gene expression profiling was hybridized on a Whole Human Genome Agilent 4X44K
Oligonucleotide Microarray. Briefly, 500 ng of mRNA were amplified then fluorescence-labeled
with Agilent’s Low RNA Input Fluorescence Linear Amplification kit (Agilent Technologies). The
sample was labeled with Cy5 and the reference Universal Human Reference RNA (Stratagene,
La Jolla, CA, USA) was labeled with Cy3. cRNA product was hybridized overnight following the
manufacturer’s instructions.
Washing steps for all platforms were performed using corresponding buffers at recommended
temperatures in an ozone-free environment. Scanning was carried out immediately using the
Agilent G2565AA Microarray Scanner System (Agilent Technologies) and data were collected
with Feature Extraction v9.5 software (Agilent Technologies).
Gene expression profiling. Microarray background subtraction was carried out using the
normexp tool available in Bioconductor’s limma package (http://www.bioconductor.org). To
normalize the dataset, loess within-array normalization and quantile between-array
normalization were performed. Differentially expressed genes were identified by fitting linear
models with the limma package (3). To take the multiple hypotheses testing into account, pvalues were adjusted using the Benjamini & Hochberg False Discovery Rate (FDR) correction.
Those genes with FDR<0.01 were defined as being differentially expressed between controls
and tumors.
RT quantitative PCR
One endogenous gene, with only minor variations in the studied series (let7a), and two small
endogenous RNAs (RNU44 and RNU48) were used (5), following the manufacturer’s
recommendations. Ct values were exported using SDS software (SDS 2.3) and the data were
analyzed with Real-Time StatMiner software (INTEGROMICSTM; www.Integromics.com). ΔCT
2
and RQ (2-ΔΔCT) were calculated using reactive lymph nodes and tonsils as the calibrator. A ttest was performed and miRNAs with FDR<0.05 were considered significant (6).
Results
Validation of microRNA expression by RT-PCR
Nineteen miRNAs were selected to confirm their expression by qPCR in MCL cases and reactive
lymphoid tissue on the basis of their statistical significance, and/or their potential role in MCL
pathogenesis (Supplementary text Table 1). Five miRNAs (RNU44, RNU48, let-7a, hsa-let7d
and hsa-miR-320) were tested as endogenous controls and the most appropriate ones (RNU44,
RNU48 and let-7a) were selected for analysis. Three out of the 19 miRNAs analyzed (miR-198,
miR-370 and miR-617) did not amplify efficiently (probably because of their low basal
expression). The significance of 13 miRNAs was confirmed (FDR<0.05), and three miRNAs
showed a similar tendency to that obtained in the microarray analysis, but their results were
not statistically significant. Thus, the majority of miRNA results obtained from the array were
confirmed by qPCR. Additional miRNAs have recently been confirmed by other groups (7).
MCL gene expression profiling
The gene expression profile in this MCL series confirmed the pathological features of this
lymphoma, with the distinctive signature including CYCLIN D1, SOX11, BMI1 and CDK4
overexpression, together with downregulation of a set of germinal center (GC) markers
including LMO2, GCET2, CD10, BCL6 and others (Supplementary text Figure 1).
3
n-fold change in
Downregulated miRs qPCR
FDR qPCR
FDR array
hsa-miR-31
0.065
<0.001
<0.001
hsa-miR-50
0.488
<0.001
<0.001
hsa-miR-24
0.525
0.004
<0.001
hsa-miR-26a
0.613
0.008
0.027
hsa-miR-200b
0.230
0.009
<0.001
hsa-miR-203
0.718
0.009
<0.001
hsa-miR-7
0.663
0.032
<0.001
hsa-miR-126
0.506
0.038
<0.001
hsa-miR-1
0.356
0.038
<0.001
hsa-miR-335
0.674
0.059
<0.001
hsa-miR-132
0.710
0.071
0.027
hsa-miR-497
0.720
0.118
<0.001
hsa-miR-363
3.503
<0.001
<0.001
hsa-miR-106b
2.068
<0.001
<0.001
hsa-miR-182
4.656
0.002
0.007
hsa-miR-181c
1.558
0.032
0.017
hsa-miR-198
ND
ND
<0.001
hsa-miR-370
ND
ND
<0.001
hsa-miR-617
ND
ND
<0.001
4
Supplementary text Table 1: RT-quantitative PCR confirmation. miRNAs used in qPCR: t-test
comparing RQ cases vs. RQ controls.
FDRs resulting from qPCR and array analysis comparison of MCL cases and reactive reactive
lymphoid tissue controls are also shown. Significance was confirmed for 13 out of 19 miRNAs
in qPCR. Three of the 19 did not have a significant FDR, but showed the same tendency
towards downregulation. Three of the 19 miRNAs did not amplify efficiently. ND: no data.
5
Supplementary text Figure 1. Gene expression profile of MCL cases. Heatmap of significant
genes (FDR<0.05). Genes downregulated and upregulated with respect to reactive lymphoid
tissue are represented in green and red, respectively. Established features of MCL disease are
confirmed, such as upregulation of Cyclin D1, SOX11 and CDK4, and downregulation of BCL6
and other germinal center markers, such as LMO2 and GCET2 (Stanford cluster).
6
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