Supplementary material:
Gene expression profiling of immunomagnetically separated cells
directly from stabilized whole blood for multicenter clinical trials
Letzkus M et al
Methods
Magnetic bead-based cell sorting
MACS® Cell Separation Technology is based on small superparamagnetic microbeads
that are bound to a highly specific antibody against a particular cell marker, thus allowing
for magnetic labeling of individual cell types. Separation occurs in a MACS Column
which induces a high-gradient magnetic fields (~ 0.6 Tesla) when placed in a MACS®
Separator.
The desired cells, collected in K2-EDTA anticoagulated whole blood samples, were
magnetically labeled with Whole Blood MicroBeads (ø 50 nm) according to the
manufacturer’s
recommendations
(Miltenyi
Biotec
GmbH,
Bergisch
Gladbach,
Germany). The cell suspension was then applied to an autoMACS Pro Separator
(Miltenyi Biotec GmbH) for fully automated separation, yielding the unlabeled negative
fraction, which was discarded, and the magnetically labeled positive fraction.. No
additional whole blood sample preparation such as density gradient centrifugation or
erythrocyte lysis was required.
The collected positive cell fraction was pelleted at 300xg for 5 minutes. One aliquot of
the final cell pellet from selected samples was used for FACS analysis and the
remaining cell pellet was lysed directly in 350 µL RLT buffer containing 1% betamercaptoethanol from the RNA extraction kit (Qiagen, Hilden, Germany).
Total RNA extraction
The total RNA from the sorted and lysed cells was isolated with the RNeasy Mini Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. After
ethanol was added to the sorted cells to adjust binding conditions, the lysate was
applied to an RNeasy spin column where RNA was selectively bound to the silica-gel
membrane, contaminants including small RNA passed through. After an on-column
DNase digestion, the remaining contaminants were removed in three efficient wash
steps and the RNA was then eluted.
The total RNA from whole blood was isolated with the PAXgene Blood RNA Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s procedure. First, the nucleic acids in
the PAXgene Blood RNA Tube were precipitated, washed and incubated in optimized
buffers together with Proteinase K to digest proteins. An additional centrifugation (4400
g for 10 minutes) removed residual cell debris. After ethanol was added to adjust binding
conditions, the lysate was applied to a PAXgene RNA spin column where RNA was
selectively bound to the silica-gel membrane as contaminants passed through. After an
on-column DNase digestion, the remaining contaminants were removed in three efficient
wash steps and the RNA was then eluted.
The total RNA was quantified by its absorbance at λ = 260 nm (A260nm) on a Nanodrop
spectrophotometer (Thermo Fisher Scientific Inc., MA, USA) and the purity was
estimated from its A260nm/A280nm ratio. RNA integrity was assessed on an Agilent
Bioanalyzer 2100 system using the Agilent RNA Pico Chip Kit (Agilent Technologies,
Inc., Santa Clara, California, USA) or on a Caliper LabChip GX system (PerkinElmer,
Waltham, Massachusetts, USA). RNA quality is reported as RNA integrity number (RIN,
(Schroeder, Mueller et al. 2006)).
Supplementary Table 1: provided in separate file
Supplementary Table 2: Hematology
WBC
RBC
HGB
HCT
PLT
N
mean
female
S.D.
CV%
median
10
6.36
2.34
36.8
5.48
10
4.80
0.46
9.5
4.74
10
14.5
1.9
13.4
13.6
10
45.0
8.3
18.4
42.4
10
227
73
32.3
227
N
mean
S.D.
CV%
median
13
5.53
1.18
21.4
5.13
13
5.50
0.87
15.9
5.24
13
16.5
2.4
14.5
15.6
13
51.2
9.2
18.0
47.1
13
179
56
31.2
181
male
Neut
count
%
10
10
3.71
58.1
1.62
10.0
43.8
17.1
3.05
61.1
13
3.17
1.01
31.7
3.0
13
56.7
7.8
13.8
57.7
Lymph
count
%
10
10
2.07
32.5
0.96
9.0
46.3
27.8
1.60
30.9
13
1.67
0.32
19.5
1.6
13
30.8
5.7
18.4
31.7
Mono
count
%
10
10
0.28
4.6
0.07
0.8
25.3
17.4
0.29
5.0
13
0.32
0.09
27.4
0.3
13
5.8
1.2
20.4
5.6
Eos
count
%
10
10
0.11
1.7
0.07
0.9
62.3
51.3
0.1
1.6
13
0.18
0.09
54.0
0.1
13
3.2
1.7
53.9
2.8
Baso
count
%
9
9
0.05
1.1
0.03
0.8
52.2
74.3
0.05
0.7
11
0.07
0.04
47.4
0.1
11
1.3
0.5
37.3
1.2
Hematology of blood samples of a subset of subjects (10 females, 13 males). WBC =
White blood cells (x 1E3 cells / μL); RBC = Red blood cells (x 1E6 cells / μL); HGB =
Hemoglobin (g / dL); HCT = Hematocrit (%); PLT = Platelets (x 1E3 cells / μL); Neut =
Neutrophil cells (x 1E3 cells / μL or %); Lymph = Lymphocytes (x 1E3 cells / μL or %);
Mono = Monocytes (x 1E3 cells / μL or %); Eos = Eosinophil cells (x 1E3 cells / μL or
%); Baso = Basophil cells (x 1E3 cells / μL or %). N, number of samples; mean, mean
count of cells x1E3 per µL blood volume; S.D., standard deviation; CV, coefficient of
variation; median, median count of cells x1E3 per µL blood volume.
Supplementary Table 3: Enrichment of cell types following MACS cell sorting
Cell type
CD3
CD4
CD8
CD15
CD19
CD45
Mean purity
(%)
97.6
94.6
95.7
98.8
86.1
91.6
S.D.
CV%
N
2.4
7.1
4.2
1.1
8.5
5.1
2.5
7.5
4.3
1.1
9.8
5.6
5
5
5
5
5
5
Cell purities after cell sorting assessed by flow cytometry (exemplary). Mean purity (%),
number of cells with desired characteristic vs all cells in sample preparation; S.D.,
standard deviation; CV, coefficient of variation; N, number of samples
Supplementary Figure 1: Cell sorting experiments - RNA yield and quality
Supplementary Figure 1: RNA quality and yield
(A) The RNA quality of the whole blood and sorted cell samples. RIN = RNA Integrity
Number.
(B) Total RNA yields from 6 mL whole blood. The total RNA was quantified by its
absorbance at λ = 260 nm. Yields were corrected for the different blood collection
volumes.
Samples from various stages of the project were considered for this analysis and do not
in all cases correspond to the number of samples used in the expression analysis.
Whiskers show the minimum to maximum range. The top and the bottom of the box are
the 75th and the 25th percentiles, respectively. The line inside the box represents the
median.
Supplementary Figure 2: Experimental procedures do not activate cells
Supplementary Figure 2: Visualization of the influence of storage temperature on cell
activation signatures.
(A) Principal component analysis (PCA) of CD14+ samples, stored in EDTA at
room temperature for up to 24 hours. Based on 503 selected cell activation
genes, a separation of sample groups by time point is notable.
(B) PCA of CD14+ samples, stored in EDTA at 4oC for up to 168 hours. No
separation by time point group is notable.
Each sphere represents a single sample. The x- and the y-axis (principal
components 1 and 2, respectively) are labeled. The z-axis is not labelled for
graphical reasons.
Reference for supplementary material
Schroeder, A., O. Mueller, et al. (2006). "The RIN: an RNA integrity number for assigning integrity values
to RNA measurements." BMC Mol Biol 7: 3.