Introduction
Materials and methods
Tissue and sample preparation for cDNA microarray
cDNA microarrays
Sample preparation for quantitative real time PCR
Bovine Samples
Human samples
Quantitative real-time PCR
Gene name | Gene symbol | NCBI ref. seq. | Forward primer | Reverse primer |
---|---|---|---|---|
Glyceraldehyde-3-phosphate dehydrogenase | GAPDH | NM_001034034.1 | TGCCGCCTGGAGAAACC | CGCCTGCTTCACCACCTT |
Aggrecan | ACAN | NM_173981.2 | GGGAGGAGACGACTGCAATC | CCCATTCCGTCTTGTTTTCTG |
Versican | VCAN | NM_181035.2 | GCTGCATGCCGCCTATG | TCCGTAGGTCCGGACTCCTT |
Collagen, type II, alpha 1 | COL2A1 | NM_001001135.2 | CGGGCTGAGGGCAACA | CGTGCAGCCATCCTTCAGA |
Synaptosomal associated protein 25 | SNAP25 | NM_001076246.1 | GGCTTCATCCGCAGGGTAA | GCTCCAGGTTTTCATCCATTTC |
Keratin 8 | KRT8 | NM_001033610.1 | ACCAGGAGCTCATGAATGTCAA | TCGCCCTCCAGCAGCTT |
Keratin 18 | KRT18 | XM_582930.4 | TTGAGCTGCTCCATCTGCAT | AAGGCCAGCTTGGAGAACAG |
Keratin 19 | KRT19 | XM_875997.3 | CGGTGCCACCATTGAGAACT | CAAACTTGGTGCGGAAGTCA |
N-Cadherin | CDH2 | XM_001250829.2 | GCCATCAAGCCAGTTGGAA | TGCAGATCGAACCGGGTACT |
Sclerostin domain containing 1 | SOSTDC1 | NM_001046265.1 | GTTCAAGTAGGCTGCCGAGAA | GCACTGGCCGTCTGAGATG |
Integrin-binding sialoprotein | IBSP | NM_174084.2 | GACAGCTATGATGGTCAAGATTACTACA | TGGGTGAACTCATCCCAGTCT |
Tenomodulin | TNMD | NM_001099948.1 | TCTGGCGTGACGGGTCTT | AAAAAAGGCATTGAACAAAACGA |
Brain attached signalling protein 1 | BASP1 | NM_174780.3 | TTGTGGATGAATGCCAACTTTC | AAAAATGGAGTATTGGCATCAAGAT |
TNF, alpha-induced protein 6 | TNFAIP6 | NM_001007813.1 | AAGCAGCAGGCGTCTACCA | CACACCGCCTTCGCTTCT |
Forkhead box F1 | FOXF1 | XM_603148.4 | TCCCTCCCCACCTCAGAAGT | TGGCTTCAGAAATGCAAGTTACTC |
Forkhead box F2 | FOXF2 | CK941878.1 | TGCGTGGTAAGTTTTCACCATCT | CCCCCGGTGAGGTAATGC |
Aquaporin 1 | AQP1 | NM_174702.3 | ACCAGGAGGCCCTTAATGGT | CTGTTAAATGGACTAGAAGCGAAATG |
Fibulin 1 | FBLN1 | NM_001098029.1 | GCAGCGCAGCCAAGTCAT | AGATATGTCTGGGTGCTACAAACG |
T, brachyury homolog (mouse) | T | XM_864890.2 | ACTTCGTGGCGGCTGACA | GCACCCACTCCCCATTCA |
Gene name | Gene symbol | NCBI ref. seq. | Forward primer | Reverse primer |
---|---|---|---|---|
Actin, beta | ACTB | NM_001101.3 | CGAGAAGATGACCCAGATCATG | ACAGCCTGGATAGCAACGTACA |
Aggrecan | ACAN | NM_001135.2 | TCTACCGCTGCGAGGTGAT | TGTAATGGAACACGATGCCTTT |
Versican | VCAN | NM_004385.4 | GCCTTTCCTATCACCTCGAGAA | CACGGCAACCCAAAATGACT |
Collagen, type II, alpha 1 | COL2A1 | NM_001844.4 | GGAAGAGTGGAGACTACTGGATTGAC | TCCATGTTGCAGAAAACCTTCA |
Synaptosomal associated protein 25 | SNAP25 | NM_003081.2 | CAATGAGCTGGAGGAGATGCA | TGCTTTCCAGCGACTCATCA |
Keratin 8 | KRT8 | NM_002273.3 | CACATCTGTGGTGCTGTCCAT | GCCTTGACCTCAGCAATGATG |
Keratin 18 | KRT18 | NM_000224.2 | GCCTACAAGCCCAGATTGC | GGCGAGGTCCTGAGATTTGG |
Keratin 19 | KRT19 | NM_002276.4 | CGCAGGGTGCTGGATGAG | AGGTAGGCCAGCTCTTCCTT |
N-Cadherin | CDH2 | NM_001792.3 | AGCCTGGAACGCAGTGTAC | GCGAACCGTCCAGTAGGAT |
Integrin-binding sialoprotein | IBSP | NM_004967.3 | CCAGAGGAAGCAATCAC | GCACAGGCCATTCCCAA |
Tenomodulin | TNMD | NM_022144.2 | CAGTGGGTGGTCCCTCAAG | GTCATTTATTGGAAGTTCTTCCTCACTTG |
Brain attached signalling protein 1 | BASP1 | NM_006317.3 | GCGGAGCCCGAGAAGAC | GGCCTCAGCAGCTTTGG |
Forkhead box F1 | FOXF1 | NM_001451.2 | AAGCCGCCCTATTCCTACATC | GCGCTTGGTGGGTGAACT |
Fibulin 1 | FBLN1 | NM_006487.2 | CCTTCGAGTGCCCTGAGAACTA | ACCGATGGCCTCATGCA |
T, brachyury homolog (mouse) | T | NM_003181.2 | CAATGAGATGATCGTGACCAAGA | GCCAGACACGTTCACCTTCA |
Results
Principal component analysis
Microarray identification of cell-specific 'marker' genes
Gene description | Gene symbol | AC mean | AF mean | NP mean | Fold change (NP/AC) | Fold change (NP/AF) | Fold change (NP/AC&AF) |
---|---|---|---|---|---|---|---|
collagen, type I, alpha 1 | COL1A1 | 21.11 | 2716.16 | 260.02 |
12.32
|
-10.45
|
-6.77
|
collagen, type I, alpha 2 | COL1A2 | 1009.68 | 15436.11 | 1330.31 |
1.32
|
-11.60
|
-6.51
|
collagen, type II, alpha 1 | COL2A1 | 13485.23 | 13260.79 | 4436.71 |
-3.04
|
-2.99
|
-3.12
|
SRY-box 9 | SOX9 | 49.26 | 8.47 | 60.38 |
1.23
|
7.13
|
1.86
|
aggrecan | ACAN | 2852.28 | 168.13 | 2467.99 |
-1.16
|
14.68
|
1.09
|
versican | VCAN | 21.36 | 1109.99 | 1864.91 |
87.30
|
1.68
|
2.59
|
Gene description | Gene symbol | AC mean | AF mean | NP mean | Fold change (NP/AC) | Fold change (NP/AF) | Fold change (NP/AC&AF) |
---|---|---|---|---|---|---|---|
matrix metallopeptidase 2 | MMP2 | 200.20 | 155.41 | 121.53 |
-1.65
|
-1.28
|
-1.56
|
solute carrier family 2, member 1 | SLC2A1 | 1200.57 | 1211.32 | 1281.16 |
1.07
|
1.06
|
1.04
|
hypoxia-inducible factor 1, alpha | HIF1A | 3985.62 | 7783.65 | 2552.07 |
-1.56
|
-3.05
|
-2.50
|
vascular endothelial growth factor | VEGF | 1874.33 | 5661.88 | 2233.09 |
1.19
|
-2.54
|
-1.86
|
CD24 molecule | CD24 | 521.84 | 44.59 | 86.75 |
-6.02
|
1.95
|
-3.42
|
keratin 19 | KRT19 | 0.91 | 0.65 | 32.59 |
35.81
|
50.47
|
19.88
|
glypican 3 | GPC3 | 4.24 | 37.63 | 13.28 |
3.13
|
-2.83
|
-1.78
|
annexin A3 | ANXA3 | 7.64 | 63.66 | 8.26 |
1.08
|
-7.71
|
-7.44
|
pleiotrophin | PTN | 208.08 | 54.11 | 1.10 |
-189.18
|
-49.19
|
-152.58
|
vimentin | VIM | 4625.18 | 4749.33 | 3508.86 |
-1.32
|
-1.35
|
-1.35
|
cartilage oligomeric matrix protein | COMP | 6053.50 | 2986.89 | 2786.96 |
-2.17
|
-1.07
|
-1.78
|
matrix Gla protein | MGP | 8480.52 | 7742.10 | 2865.30 |
-2.96
|
-2.70
|
-3.21
|
alpha-2-macroglobulin | A2M | 9.49 | 26.62 | 11.37 |
1.20
|
-2.34
|
-1.77
|
annexin A4 | ANXA4 | 1223.21 | 2318.40 | 1273.29 |
1.04
|
-1.82
|
-1.55
|
desmocollin 2 | DSC2 | 1.88 | 140.72 | 68.18 |
36.30
|
-2.06
|
-1.22
|
keratin 18 | KRT18 | 0.01 | 0.04 | 94.10 |
15990.45
|
2647.60
|
1860.77
|
neural cell adhesion molecule 1 | NCAM1 | 0.76 | 0.94 | 12.64 |
16.55
|
13.43
|
10.06
|
Gene description | Gene symbol | AC mean | AF mean | AC&AF mean | NP mean | Fold change (NP/AC&AF) |
---|---|---|---|---|---|---|
keratin 8
|
KRT8
|
0.09
|
0.39
|
0.28
|
714.79
|
2544.79
|
keratin 18
|
KRT18
|
0.01
|
0.04
|
0.05
|
94.10
|
1860.77
|
ras homolog gene family, member B | RHOB | 0.24 | 0.04 | 0.40 | 383.01 |
968.68
|
hypothetical protein BC012029 | LOC152573 | 0.05 | 0.18 | 0.26 | 230.02 |
897.02
|
cadherin 2, type 1, N-cadherin
|
CDH2
|
0.09
|
0.22
|
0.21
|
180.47
|
840.69
|
Kruppel-like factor 6 | KLF6 | 0.13 | 0.03 | 0.12 | 55.84 |
456.37
|
plakophilin 2 | PKP2 | 0.13 | 0.48 | 0.37 | 77.71 |
212.23
|
related RAS viral (r-ras) oncogene homolog | RRAS | 0.74 | 0.39 | 0.61 | 93.40 |
152.49
|
synaptosomal-associated protein, 25 kDa
|
SNAP25
|
0.55
|
0.81
|
0.69
|
94.22
|
136.76
|
sclerostin domain containing 1
|
SOSTDC1
|
0.28
|
0.53
|
0.68
|
79.27
|
116.29
|
optineurin | OPTN | 0.22 | 0.36 | 0.80 | 88.35 |
109.86
|
tropomyosin 2 (beta)
|
TPM2
|
18.99
|
94.17
|
78.02
|
0.74
|
-104.73
|
integrin, alpha 9 | ITGA9 | 38.74 | 62.19 | 66.25 | 0.50 |
-131.50
|
pleiotrophin (heparin binding growth factor 8)
|
PTN
|
208.08
|
54.11
|
167.82
|
1.10
|
-152.58
|
Tissue factor pathway inhibitor 2 | TFPI2 | 1080.55 | 5089.17 | 4138.76 | 26.91 |
-153.83
|
guanine nucleotide binding protein (G protein), gamma 11 | GNG11 | 0.57 | 331.31 | 298.19 | 1.65 |
-181.06
|
podocalyxin-like | PODXL | 0.50 | 297.24 | 208.74 | 1.14 |
-182.62
|
proteoglycan 4 | PRG4 | 917.06 | 27.09 | 722.28 | 3.25 |
-222.06
|
a disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 4 | ADAMTS4 | 115.72 | 2187.60 | 1561.34 | 5.25 |
-297.16
|
phenylalanine hydroxylase | PAH | 195.83 | 0.24 | 98.50 | 0.32 |
-307.92
|
interferon, gamma-inducible protein 16 | IFI16 | 0.28 | 61.31 | 67.55 | 0.20 |
-337.80
|
retinoic acid receptor responder 1 | RARRES1 | 974.75 | 156.02 | 671.46 | 1.98 |
-338.73
|
regulator of G-protein signalling 5 | RGS5 | 5.68 | 436.91 | 264.83 | 0.67 |
-393.62
|
integrin-binding sialoprotein
|
IBSP
|
300.90
|
0.06
|
159.69
|
0.37
|
-433.73
|
complement factor H | CFH | 1.72 | 60.74 | 86.66 | 0.12 |
-729.93
|
fibulin 1
|
FBLN1
|
199.29
|
0.22
|
108.88
|
0.13
|
-826.03
|
Serpin peptidase inhibitor, clade F, member 1 | SERPINF1 | 17.15 | 512.43 | 281.58 | 0.34 |
-830.27
|
Keratocan | KERA | 0.05 | 346.47 | 341.38 | 0.38 |
-893.66
|
collagen, type X, alpha 1
|
COL10A1
|
1192.84
|
7.19
|
741.87
|
0.79
|
-944.44
|
Endomucin | EMCN | 0.45 | 281.94 | 189.76 | 0.16 |
-1166.81
|
secreted frizzled-related protein 2 | SFRP2 | 9.98 | 153.89 | 106.73 | 0.08 |
-1391.84
|
peptidase inhibitor 15 | PI15 | 478.32 | 83.74 | 308.94 | 0.10 |
-3231.26
|
myosin, heavy chain 11, smooth muscle | MYH11 | 1.15 | 462.12 | 263.70 | 0.03 |
-7708.90
|
chemokine (C-X-C motif) ligand 1 | CXCL1 | 3.14 | 549.84 | 492.06 | 0.02 |
-28150.12
|
Gene description | Gene symbol | AC mean | AF mean | NP mean | Fold change (NP/AC) |
---|---|---|---|---|---|
brain abundant, membrane attached signal protein 1 | BASP1 | 0.02 | 57.19 | 384.21 | 16167.90 |
keratin 18
|
KRT18
|
0.01
|
0.04
|
94.10
|
15990.45
|
Tenomodulin
|
TNMD
|
0.01
|
241.03
|
102.36
|
15087.56
|
TNF, alpha-induced protein 6
|
TNFAIP6
|
0.01
|
304.98
|
121.88
|
8205.96
|
keratin 8
|
KRT8
|
0.09
|
0.39
|
714.79
|
7607.92
|
hypothetical protein BC012029 | LOC152573 | 0.05 | 0.18 | 230.02 |
4635.12
|
TNF, alpha-induced protein 6
|
TNFAIP6
|
0.19
|
3649.85
|
508.71
|
2648.51
|
SH3 domain binding glutamic acid-rich protein | SH3BGR | 0.08 | 20.83 | 172.70 |
2230.52
|
cadherin 2, type 1, N-cadherin (neuronal)
|
CDH2
|
0.09
|
0.22
|
180.47
|
1941.17
|
chordin | CHRD | 0.03 | 1.29 | 57.29 | 1671.45 |
Rat sarcoma (ras) homolog gene family, member B | RHOB | 0.24 | 0.04 | 383.01 |
1627.81
|
homeobox B8 | HOXB8 | 0.08 | 9.11 | 119.75 |
1591.12
|
Rho GTPase activating protein 27 | ARHGAP27 | 0.08 | 0.72 | 89.24 |
1072.84
|
forkhead box F1
|
FOXF1
|
0.43
|
703.54
|
457.76
|
1054.86
|
plakophilin 2 | PKP2 | 0.13 | 0.48 | 77.71 |
603.25
|
homeobox B6 | HOXB6 | 0.11 | 58.21 | 61.73 |
580.12
|
adaptor-related protein complex 2, mu 1 subunit | AP2M1 | 0.55 | 350.92 | 305.70 |
557.26
|
transketolase-like 1 | TKTL1 | 0.89 | 4.59 | 478.18 |
536.27
|
cytochrome b-245, alpha polypeptide | CYBA | 0.16 | 0.77 | 83.63 |
513.67
|
phosphatidylethanolamine-binding protein 4 | PEBP4 | 0.84 | 113.44 | 429.31 |
511.40
|
forkhead box F2
|
FOXF2
|
0.60
|
416.30
|
283.89
|
470.91
|
Kruppel-like factor 6 | KLF6 | 0.13 | 0.03 | 55.84 |
444.91
|
optineurin | OPTN | 0.22 | 0.36 | 88.35 |
407.25
|
aquaporin 1 (Colton blood group)
|
AQP1
|
0.42
|
5.55
|
144.13
|
346.12
|
RAB3B, member RAS oncogene family | RAB3B | 0.18 | 0.27 | 58.47 |
333.33
|
similar to sushi domain containing 2 | SUSD2 | 1.05 | 34.26 | 300.30 |
286.96
|
sclerostin domain containing 1
|
SOSTDC1
|
0.28
|
0.53
|
79.27
|
286.23
|
CD36 molecule (thrombospondin receptor) | CD36 | 0.27 | 96.74 | 71.61 |
262.65
|
capping protein (actin filament), gelsolin-like | CAPG | 0.21 | 12.70 | 53.11 |
257.62
|
similar to zinc finger homeodomain 4 | ZFHX4 | 0.23 | 160.03 | 55.13 |
241.22
|
neurotrophic tyrosine kinase, receptor, type 2 | NTRK2 | 0.62 | 248.28 | 106.60 |
172.40
|
synaptosomal-associated protein, 25 kDa
|
SNAP25
|
0.55
|
0.81
|
94.22
|
171.81
|
lectin, galactoside-binding, soluble, 1 (galectin 1) | LGALS1 | 0.57 | 81.20 | 88.20 |
155.33
|
testis derived transcript (3 Lin11, Isl-1 & Mec-3 (LIM) domains) | TES | 0.38 | 32.63 | 55.66 |
145.10
|
Related-rat sarcoma viral (r-ras) oncogene homolog | RRAS | 0.74 | 0.39 | 93.40 |
125.71
|
sorting nexin family member 30 | SNX30 | 0.74 | 31.31 | 89.00 |
120.18
|
vanin 1 | VNN1 | 0.69 | 67.28 | 82.51 |
119.73
|
collagen, type XVIII, alpha 1 | COL18A1 | 1.02 | 102.46 | 116.62 |
114.48
|
transmembrane protein 100 | TMEM100 | 1.05 | 1.80 | 116.48 |
110.68
|
macrophage migration inhibitory factor | MIF | 3.47 | 1.70 | 368.05 |
105.96
|
ectodermal-neural cortex | ENC1 | 0.74 | 53.63 | 75.21 |
101.74
|
pleiotrophin
|
PTN
|
208.08
|
54.11
|
1.10
|
-189.18
|
cytokine-like 1 | CYTL1 | 11388.79 | 19.33 | 29.48 |
-386.29
|
retinoic acid receptor responder 1 | RARRES1 | 974.75 | 156.02 | 1.98 |
-491.73
|
phenylalanine hydroxylase | PAH | 195.83 | 0.24 | 0.32 |
-612.20
|
integrin-binding sialoprotein
|
IBSP
|
300.90
|
0.06
|
0.37
|
-817.30
|
fibulin 1
|
FBLN1
|
199.29
|
0.22
|
0.13
|
-1511.93
|
collagen, type X, alpha 1
|
COL10A1
|
1192.84
|
7.19
|
0.79
|
-1518.56
|
peptidase inhibitor 15 | PI15 | 478.32 | 83.74 | 0.10 |
-5002.83
|