Identification of Importin 8 (IPO8) as the most accurate housekeeping gene
for the clinicopathological analysis of lung specimens
Paul A. Nguewa1 , Jackeline Agorreta 1 , David Blanco 1 , Maria Dolores Lozano2,
Javier Gomez-Roman3, Blas A. Sanchez 4 , Iñaki Valles1, Maria J. Pajares 1, Ruben
Pio1, Maria Jose Rodriguez 4 , Luis M. Montuenga 1#*, Alfonso Calvo 1#
1
Division of Oncology, Center for Applied Medical Research (CIMA), University
of Navarra. Avda. Pio XII, 55. 31008 Pamplona, Spain.
2
Department of Pathology. University Hospital of Navarra. Avda. Pio XII, 36. 31008
Pamplona, Spain
3
Department of Anatomical Pathology, Marqués de Valdecilla University
Hospital, Medical Faculty, University of Cantabria, Santander, Spain.
4
Research Department. Ingenasa
#: Contributed equally to this paper
Email addresses:
PAN: panguewa@unav.es
JA: jagorreta@unav.es
DB: dblanco@unav.es
MDL: mdlozano@unav.es
JG-R: apagrj@humv.es
BAS: bsanchez@ingenasa.es
IV: ivalles@unav.es
MJP: mpajares@unav.es
RP: rpio@unav.es
MJR: mjrodriguez@ingenasa.es
LM*: lmontuenga@unav.es
AC: acalvo@unav.es
* Corresponding author:
“Online Data Supplement”
Sources of support:
This work has been funded by “UTE project CIMA”, ISCIII-RETIC RD06/0020 grant;
Cenit project ONCNOSIS, Government of Navarra. P.A.N. was supported by a Spanish
Torres-Quevedo fellowship (PTQ05-01-01084) and J.A. by a fellowship from the
“Instituto de Salud Carlos III”, Ministry of Health, Spain.
Supplemental Table 1: Clinical characteristics of patients with lung cancer
SET A
SET B
SET C
No
Sex
Age (y)
Diagnosis
Stage
Smoking
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
F
F
M
M
F
F
M
M
M
F
M
M
F
M
M
M
M
M
M
M
M
M
M
F
M
M
M
M
F
M
M
M
M
M
M
M
M
37
52
75
58
71
54
55
60
70
73
50
58
71
60
69
54
53
67
52
71
48
75
52
59
68
73
74
78
66
70
66
75
63
60
87
75
63
Squamous Cell Carcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Squamous Cell Carcinoma
Squamous Cell Carcinoma
Adenocarcinoma
IB
IB
IA
IA
IB
IIIB
IA
IA
IIB
IIB
IB
IA
IB
IIIB
IB
IB
IB
IB
IIIA
IA
IIA
IB
IB
IB
IIIA
IIIA
IIIA
IB
IB
IB
IB
IIIA
IB
IB
IB
IIIA
IB
Yes
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
U
Yes
Yes
Yes
U
Yes
U
Yes
U: Unknown
1
Supplemental Table 2 Absolute values of ∆Ct (|∆Ct|=|Ct Normal-Ct Tumor|) of
GAPDH and HPRT1 in paired lung clinical samples (Set A). Mean and Standard error
of mean (SEM) were calculated.
SET A
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
GAPDH
|∆Ct|
2.09
3.14
2.22
2.21
0.45*
1.17
1.97
1.12
4.99
0.83*
3.55
2.81
2.20
1.84
3.43
HPRT1
|∆Ct|
1.23
2.02
1.24
1.19
0.45*
1.59
2.22
1.75
4.37
0.06*
2.42
2.56
1.88
1.69
2.50
Mean
SEM
2.27
0.31
1.81
0.26
*: Negative values
2
Supplemental Table 3 ANOVA test of three lung cancer patient microarrays (HuGene-FL, HG-U95A, HG-U133A). The raw datasets are
publicly available at the website: www.affymetrix.com/products/arrays/index.affx. (*) p-values <0.05 were considered statistically significant.
GAPDH
HPRT1
HG-U95A (Tumor vs. Normal)
Probe
p-value
35905_s_at
0.000000 (*)
AFFX-HUMGAPDH/M33197_3_at
0.000000 (*)
AFFX-HUMGAPDH/M33197_5_at
0.000000 (*)
AFFX-HUMGAPDH/M33197_5_st
0.004315 (*)
AFFX-HUMGAPDH/M33197_M_at 0.000000 (*)
AFFX-HUMGAPDH/M33197_M_st 0.048685 (*)
AFFX-HUMGAPDH/M33197_3_st
> 0.05
37640_at
0.000025 (*)
3
HuGene-FL (Tumor vs. Normal)
Probe
p-value
AFFX-HUMGAPDH/M33197_3_at
0.000106 (*)
AFFX-HUMGAPDH/M33197_3_st
0.040188 (*)
AFFX-HUMGAPDH/M33197_5_at
0.000010 (*)
AFFX-HUMGAPDH/M33197_5_st
0.043371 (*)
AFFX-HUMGAPDH/M33197_M_at
0.000178 (*)
AFFX-HUMGAPDH/M33197_M_st
> 0.05
X01677_f_at
0.000010 (*)
M31642_at
0.009939 (*)
HG-U133A (Tumor vs. Normal)
Probe
p-value
212581_x_at
0.000014 (*)
213453_x_at
0.000015 (*)
217398_x_at
0.000015 (*)
AFFX-HUMGAPDH/M33197_3_at
0.000075 (*)
AFFX-HUMGAPDH/M33197_5_at
0.000152 (*)
AFFX-HUMGAPDH/M33197_M_at
0.000005 (*)
202854_at
0.000432 (*)
Supplemental Text 1. Correlation between endogenous control genes in lung tumor
cell lines
The four strongest correlations (**p<0.01) (See supplemental Table 4, in bold)
were found between RPLPO and ACTB (r=0.970), PPIA and ACTB (r=0.969), PPIA
and HPRT (r=0.949), and GAPDH and POLR2A (r=0.944). In addition, we also
evaluated the number of significant correlations (**p<0.01) of each gene among the
sixteen others, in lung cell lines (See supplemental Table 5). We then classified the
genes in three groups depending on the number of significant correlation (NC). The
ones with very good and good numbers of significant correlation were: PPIA and ACTB
(NC=11/16); HPRT1, GAPDH, RPLPO (NC=9/16); and 18S (NC=8/16). Those with
bad significant correlation were: UBC (NC=7/16); PGK1, POLR2A (NC=6/16), GUSB,
TBP (NC=5/16), and HMBS (NC=4/16). The last group included genes with the lowest
NC, thus indicating the worse correlations with other reference genes: YWHAZ
(NC=3/16); B2M (NC=2/16); IPO8 and TFRC (NC=1/16).
4
A
HKG
18S
ACTB
B2M
GAPDH
GUSB
HMBS
HPRT1
IPO8
PGK1
POLR2A
PPIA
RPLPO
TBP
TFRC
UBC
YWHAZ
Supplemental Table 4 Correlation between sixteen reference genes in lung cancer cell lines.
18S
ACTB
0.883(**)
B2M
0.797
0.662
GAPDH
0.882(**)
0.933(**)
0.578
GUSB
0.808(**)
0.804(**)
0.446
0.716
HMBS
0.665
0.770
0.222
0.729
0.778
HPRT1
0.828(**)
0.906(**)
0.466
0.814(**)
0.913(**)
0.919(**)
IPO8
0.574
0.363
0.239
0.498
0.657
0.288
0.388
PGK1
0.628
0.880(**)
0.423
0.807(**)
0.558
0.704
0.744
0.054
POLR2A
0.723
0.836(**)
0.317
0.944(**)
0.595
0.750
0.754
0.387
0.820(**)
PPIA
0.929(**)
0.969(**)
0.641
0.931(**)
0.869(**)
0.835(**)
0.949(**)
0.479
0.765
0.826(**)
RPLPO
0.869(**)
0.970(**)
0.653
0.933(**)
0.705
0.779
0.855(**)
0.307
0.895(**)
0.866(**)
0.937(**)
TBP
0.607
0.826(**)
0.406
0.701
0.679
0.803(**)
0.862(**)
0.052
0.714
0.644
0.831(**)
0.757
TFRC
0.248
0.263
0.289
0.110
0.285
0.523
0.424
-0.199
0.122
-0.004
0.358
0.274
0.590
UBC
0.796
0.861(**)
0.759
0.805(**)
0.580
0.648
0.697
0.201
0.798(**)
0.656
0.817(**)
0.892(**)
0.682
0.449
Data are represented as coefficients (r-value) between each two housekeeping genes. HKG: housekeeping genes. (**) p-Values are considered significant when p<0.01.
5
YWHAZ
0.802(**)
0.712
0.815(**)
0.702
0.610
0.282
0.486
0.603
0.525
0.473
0.672
0.684
0.300
-0.009
0.768
Supplemental Table 5 Number of significant correlations (NC) (**p<0.01) of each
gene over all the sixteen endogenous control genes in lung cancer cell lines.
B
Cell Lines (Pearson Correlation)
16 Genes
NC (over 16)
ACTB
11
PPIA
11
HPRT1
9
GAPDH
9
RPLPO
9
18S
8
UBC
6
PGK1
6
POLR2A
6
GUSB
5
TBP
5
HMBS
4
YWHAZ
3
B2M
2
IPO8
1
TFRC
1
6
Supplemental Text S2. Correlation between endogenous control genes in normal and
tumoral lung tissues
In Supplemental Table 6, correlations between endogenous control genes in lung
tissues are represented as coefficients (r-value). Most of endogenous control genes were
strongly correlated. The strongest correlations (**p<0.01) (Table S5, in bold) were
between IPO8 and ACTB (r=0.981), PPIA and RPLPO (r=0.969), POLR2A and 18S
(r=0.962), IPO8 and POLR2A, PPIA and HMBS, GAPDH and PGK1 (r=0.957), HPRT
and GAPDH (r=0.956), PPIA and POLR2A (r=0.954), and PPIA and PGK1 (r=0.953).
The evaluation of the numbers of significant correlation (**p<0.01) of each gene among
the sixteen others in lung tissues (See Supplemental Table 7) revealed two groups of
genes. Except for three genes, all the others exhibited very good or good NC, ranging
from 14/16, to 11/16. The best one was IPO8 (NC=14/16). PPIA, ACTB, HMBS,
POLR2A, 18S, and RPLPO had NC=13/16; HPRT, PGK1, TBP, and YWHAZ:
NC=12/16; and finally, GAPDH and UBC, NC=11/16. However, the group with the
worse numbers of significant correlation was: B2M and TFRC (NC=3/16), and GUSB
(NC=1/16).
Consequently, this analysis shows that IPO8, 18S, ACTB, POLR2A, PPIA,
HMBS, and RPLPO exhibit a strong correlation expression among them (r=0.9810.953) as well as high numbers of significant correlations (NC=14-13 over the sixteen
genes).
7
Supplemental Table 6 Correlation between sixteen reference genes in lung cancer tissues.
HKG
18S
ACTB
B2M
GAPDH
GUSB
HMBS
HPRT1
IPO8
PGK1
POLR2A
PPIA
RPLPO
TBP
TFRC
UBC
YWHAZ
18S
ACTB
0.857(**)
B2M
0.644
0.802(**)
GAPDH
0.756(**)
0.705
0.275
GUSB
0.486
0.616
0.268
0.520
HMBS
0.926(**)
0.819(**)
0.515
0.868(**)
0.627
HPRT1
0.715(**)
0.716(**)
0.320
0.956(**)
0.477
0.792(**)
IPO8
0.908(**)
0.981(**)
0.779(**)
0.722(**)
0.581
0.848(**)
0.738(**)
PGK1
0.836(**)
0.718(**)
0.281
0.957(**)
0.595
0.928(**)
0.921(**)
0.753(**)
POLR2A
0.962(**)
0.945(**)
0.702
0.817(**)
0.561
0.924(**)
0.789(**)
0.957(**)
0.846(**)
PPIA
0.922(**)
0.863(**)
0.485
0.932(**)
0.614
0.957(**)
0.889(**)
0.889(**)
0.953(**)
0.954(**)
RPLPO
0.926(**)
0.821(**)
0.514
0.924(**)
0.421
0.923(**)
0.891(**)
0.866(**)
0.921(**)
0.941(**)
0.969(**)
TBP
0.844(**)
0.721(**)
0.577
0.702
0.388
0.835(**)
0.725(**)
0.780(**)
0.757(**)
0.798(**)
0.811(**)
0.829(**)
TFRC
0.526
0.441
0.096
0.746(**)
0.336
0.533
0.829(**)
0.512
0.700
0.552
0.680
0.701
0.640
UBC
0.846(**)
0.746(**)
0.715(**)
0.649
0.170
0.759(**)
0.701
0.780(**)
0.705
0.822(**)
0.741(**)
0.823(**)
0.810(**)
0.441
Data are represented as coefficients (r-value) between each two housekeeping genes. HKG: housekeeping genes. (**) p-Values are considered significant when p<0.01.
8
YWHAZ
0.894(**)
0.824(**)
0.680
0.777(**)
0.483
0.910(**)
0.698
0.863(**)
0.777(**)
0.895(**)
0.872(**)
0.884(**)
0.829(**)
0.458
0.778(**)
Supplemental Table 7 Number of significant correlations (NC) (**p<0.01) of each
gene over all the sixteen endogenous control genes in lung cancer tissues.
Tissues (Pearson Correlation)
16 Genes
NC (over 16)
IPO8
14
18S
13
ACTB
13
HMBS
13
POLR2A
13
PPIA
13
RPLPO
13
HPRT1
12
PGK1
12
TBP
12
YWHAZ
12
GAPDH
11
UBC
10
B2M
4
TFRC
3
GUSB
1
9