PKF arrays only

Boxplots of Red, Green, Red background, and Green background

## Read PKF/20110210-3_1-4.gpr 
## Read PKF/20110210-3_2-4.gpr 
## Read PKF/20110210-3_3-4.gpr 
## Read PKF/20110210-3_4-4.gpr

One array is not like the others, so it will be excluded

## Warning in plotDensities.RGList(RG): NaNs produced

## Warning in plotDensities.RGList(RG[, -1]): NaNs produced

Background correction

## Array 1 corrected
## Array 2 corrected
## Array 3 corrected
## Array 1 corrected
## Array 2 corrected
## Array 3 corrected

Loess Normalization

Normalization between arrays (Aquantile)

Differential Gene Expression, no genes are significant

## Found unique target names:
##  ctrl pkf
##       Block Column Row            Name            ID ControlType GeneName
## 16800     1     42  64       NM_016257  A_24_P139665       false   HPCAL4
## 21478     1    198  81       NM_176890  A_23_P347029       false  TAS2R50
## 6464      1     80  25       NM_024917 A_33_P3258997       false   TRMT2B
## 4405      1    149  17       NM_000777    A_23_P8801       false   CYP3A5
## 26204     1    136  99 ENST00000395482 A_33_P3409710       false         
## 7850      1    136  30                  A_24_P392230       false         
##                                                                          TopHit
## 16800                 ref|NM_016257|ens|ENST00000372844|gb|BC030827|gb|AL136591
## 21478                             ref|NM_176890|ens|ENST00000390675|gb|BC112100
## 6464  ref|NM_024917|ens|ENST00000372931|ens|ENST00000338687|ens|ENST00000372936
## 4405                  ref|NM_000777|ens|ENST00000222982|gb|CR620792|gb|CR611286
## 26204                                                       ens|ENST00000395482
## 7850                                                                           
##                                                                                           Description
## 16800                                      Homo sapiens hippocalcin like 4 (HPCAL4), mRNA [NM_016257]
## 21478                      Homo sapiens taste receptor, type 2, member 50 (TAS2R50), mRNA [NM_176890]
## 6464  Homo sapiens TRM2 tRNA methyltransferase 2 homolog B (S. cerevisiae) (TRMT2B), mRNA [NM_024917]
## 4405    Homo sapiens cytochrome P450, family 3, subfamily A, polypeptide 5 (CYP3A5), mRNA [NM_000777]
## 26204             Zinc finger protein 713  [Source:UniProtKB/Swiss-Prot;Acc:Q8N859] [ENST00000395482]
## 7850                                                                                                 
##           logFC  AveExpr        t      P.Value adj.P.Val         B
## 16800 0.9254404 6.914536 6.350825 0.0006904733 0.9999765 -2.986137
## 21478 0.5082917 6.592153 6.048304 0.0008955324 0.9999765 -3.020188
## 6464  0.5764991 7.671875 5.937149 0.0009877645 0.9999765 -3.033680
## 4405  0.4989574 6.290596 5.657514 0.0012718390 0.9999765 -3.070199
## 26204 0.7683298 6.555933 5.432476 0.0015691914 0.9999765 -3.102528
## 7850  1.2685910 8.265459 5.267019 0.0018385822 0.9999765 -3.128144

Analysis with all arrays

## Read PKF/20110210-3_1-4.gpr 
## Read PKF/20110210-3_2-4.gpr 
## Read PKF/20110210-3_3-4.gpr 
## Read PKF/20110210-3_4-4.gpr 
## Read WNT/Wnt_1-4.gpr 
## Read WNT/Wnt_2-4.gpr 
## Read WNT/Wnt_3-4.gpr 
## Read WNT/Wnt_4-4.gpr

PKF arrays compared to WNT don’t have as good signal strength

## Warning in plotDensities.RGList(RG): NaNs produced

Background correct, Normalize within, Normalize between arrays

## Array 1 corrected
## Array 2 corrected
## Array 3 corrected
## Array 4 corrected
## Array 5 corrected
## Array 6 corrected
## Array 7 corrected
## Array 1 corrected
## Array 2 corrected
## Array 3 corrected
## Array 4 corrected
## Array 5 corrected
## Array 6 corrected
## Array 7 corrected

Differential gene expression analysis

## Found unique target names:
##  ctrl_pkf ctrl_wnt pkf wnt

Wnt array is fine

##       Block Column Row         Name           ID ControlType GeneName
## 38024     1    252 143    NM_001673 A_23_P145694       false     ASNS
## 32182     1    262 121 NM_001040619  A_23_P34915       false     ATF3
## 42139     1    111 159    NM_018948  A_23_P46470       false   ERRFI1
## 17940     1    118  68    NM_001964 A_23_P214080       false     EGR1
## 26297     1    229  99    NM_001730  A_23_P53891       false     KLF5
## 3375      1    183  13    NM_004414 A_23_P166248       false    RCAN1
##                                                                    TopHit
## 38024       ref|NM_001673|ref|NM_133436|ref|NM_183356|ens|ENST00000422745
## 32182 ref|NM_001040619|ref|NM_001674|ref|NM_001030287|ens|ENST00000366987
## 42139           ref|NM_018948|ens|ENST00000377482|gb|AK027830|gb|AK096149
## 17940               ref|NM_001964|ens|ENST00000239938|gb|M80583|gb|M62829
## 26297           ref|NM_001730|ens|ENST00000377687|gb|AK131397|gb|AF132818
## 3375        ref|NM_004414|ref|NM_203418|ref|NM_203417|ens|ENST00000443408
##                                                                                            Description
## 38024                Homo sapiens asparagine synthetase (ASNS), transcript variant 2, mRNA [NM_001673]
## 32182 Homo sapiens activating transcription factor 3 (ATF3), transcript variant 4, mRNA [NM_001040619]
## 42139                       Homo sapiens ERBB receptor feedback inhibitor 1 (ERRFI1), mRNA [NM_018948]
## 17940                                    Homo sapiens early growth response 1 (EGR1), mRNA [NM_001964]
## 26297                         Homo sapiens Kruppel-like factor 5 (intestinal) (KLF5), mRNA [NM_001730]
## 3375           Homo sapiens regulator of calcineurin 1 (RCAN1), transcript variant 1, mRNA [NM_004414]
##           logFC   AveExpr         t      P.Value    adj.P.Val        B
## 38024 -4.249590 11.346495 -62.56421 1.874039e-14 8.080480e-10 22.47485
## 32182 -4.267144  8.634658 -57.36133 4.508179e-14 9.719184e-10 21.88858
## 42139 -3.408687 10.181561 -52.82613 1.036277e-13 1.489406e-09 21.29672
## 17940 -4.542792 11.055544 -47.23067 3.210129e-13 2.806035e-09 20.43967
## 26297 -3.067099  7.589910 -46.41165 3.830059e-13 2.806035e-09 20.30060
## 3375  -3.443266 10.900700 -46.32300 3.904682e-13 2.806035e-09 20.28532

PKF array still doesn’t have significant genes

##       Block Column Row            Name            ID ControlType  GeneName
## 26204     1    136  99 ENST00000395482 A_33_P3409710       false          
## 8378      1    132  32    NM_001018072  A_23_P419714       false    BTBD11
## 17751     1    195  67       NM_002777  A_23_P142345       false     PRTN3
## 22116     1     38  84       XR_040729 A_33_P3374165       false LOC728145
## 6985      1     69  27       NM_020157 A_33_P3311830       false      OTOR
## 27493     1     95 104       XR_041259 A_33_P3395219       false  C1orf118
##                                                                          TopHit
## 26204                                                       ens|ENST00000395482
## 8378  ref|NM_001018072|ref|NM_001017523|ens|ENST00000280758|ens|ENST00000357167
## 17751                 ref|NM_002777|ens|ENST00000234347|gb|AB527757|gb|BC096183
## 22116                     ref|XR_040729|ref|XR_040728|ref|XR_040730|gb|AK090994
## 6985                  ref|NM_020157|ens|ENST00000246081|gb|AJ242552|gb|CR624240
## 27493                     ref|XR_041259|ref|XR_041257|ref|XR_041258|gb|AK075118
##                                                                                           Description
## 26204             Zinc finger protein 713  [Source:UniProtKB/Swiss-Prot;Acc:Q8N859] [ENST00000395482]
## 8378  Homo sapiens BTB (POZ) domain containing 11 (BTBD11), transcript variant a, mRNA [NM_001018072]
## 17751                                             Homo sapiens proteinase 3 (PRTN3), mRNA [NM_002777]
## 22116         PREDICTED: Homo sapiens hypothetical protein LOC728145 (LOC728145), miscRNA [XR_040729]
## 6985                                                  Homo sapiens otoraplin (OTOR), mRNA [NM_020157]
## 27493     PREDICTED: Homo sapiens chromosome 1 open reading frame 118 (C1orf118), miscRNA [XR_041259]
##           logFC  AveExpr        t      P.Value adj.P.Val         B
## 26204 0.7683298 6.496862 6.786171 4.511836e-05 0.9999821 -1.991661
## 8378  0.5591699 6.341462 5.856714 1.518209e-04 0.9999821 -2.213788
## 17751 0.6639550 6.543475 5.679206 1.939474e-04 0.9999821 -2.263860
## 22116 0.5621299 7.087566 5.515727 2.439589e-04 0.9999821 -2.312458
## 6985  1.8636474 6.987066 5.441070 2.712441e-04 0.9999821 -2.335478
## 27493 0.8203388 6.905016 5.414623 2.816782e-04 0.9999821 -2.343761

Heatmap with probes

Extracted all probes that match the probes from PKF DEG table

##            PKF_2       PKF_3      PKF_4   WNT3a_1    WNT3a_2   WNT3a_3
##   1:  0.38886916 -0.10408281 -0.1853648 -1.323171 -1.5069663 -1.800176
##   2:  0.03956187 -0.04363556 -0.1522391 -2.102709 -1.8539398 -2.159064
##   3:  0.14591395 -0.08635766  0.3556778 -2.252406 -2.0880409 -2.282818
##   4:  0.15079773 -0.25385427 -0.2636840 -1.009738 -1.3152536 -1.160213
##   5:  0.48501334  0.12014334  0.1387680 -1.260909 -1.2482922 -1.136186
##  ---                                                                  
##  97:  0.23621100  0.19614692  0.4565083 -1.761846 -1.7116754 -1.615818
##  98: -0.23267039  0.05051863  0.1969405 -2.881302 -3.0130705 -2.638797
##  99: -0.14499814  0.21891467  0.1781160 -1.182916 -0.9998219 -1.158014
## 100: -0.09811721 -0.06362834 -0.2412201 -1.637103 -1.7018866 -2.078183
## 101:  0.01451336  0.41064085  0.4247249 -1.757982 -1.7188237 -1.553344
##         WNT3a_4        Probe
##   1: -1.6161890 A_32_P231617
##   2: -2.3241334 A_23_P122924
##   3: -2.3685847 A_23_P122924
##   4: -0.9989936 A_23_P118150
##   5: -1.0774271 A_24_P250922
##  ---                        
##  97: -1.8709626   A_23_P1691
##  98: -3.1170994  A_23_P79518
##  99: -1.1331159 A_23_P374844
## 100: -2.2965098 A_23_P122924
## 101: -1.9277140   A_23_P1691
##            PKF_2       PKF_3      PKF_4   WNT3a_1    WNT3a_2   WNT3a_3
##   1:  0.38886916 -0.10408281 -0.1853648 -1.323171 -1.5069663 -1.800176
##   2:  0.03956187 -0.04363556 -0.1522391 -2.102709 -1.8539398 -2.159064
##   3:  0.14591395 -0.08635766  0.3556778 -2.252406 -2.0880409 -2.282818
##   4:  0.15079773 -0.25385427 -0.2636840 -1.009738 -1.3152536 -1.160213
##   5:  0.48501334  0.12014334  0.1387680 -1.260909 -1.2482922 -1.136186
##  ---                                                                  
##  97:  0.23621100  0.19614692  0.4565083 -1.761846 -1.7116754 -1.615818
##  98: -0.23267039  0.05051863  0.1969405 -2.881302 -3.0130705 -2.638797
##  99: -0.14499814  0.21891467  0.1781160 -1.182916 -0.9998219 -1.158014
## 100: -0.09811721 -0.06362834 -0.2412201 -1.637103 -1.7018866 -2.078183
## 101:  0.01451336  0.41064085  0.4247249 -1.757982 -1.7188237 -1.553344
##         WNT3a_4        Probe    Gene
##   1: -1.6161890 A_32_P231617  TM4SF1
##   2: -2.3241334 A_23_P122924   INHBA
##   3: -2.3685847 A_23_P122924   INHBA
##   4: -0.9989936 A_23_P118150 ARL6IP1
##   5: -1.0774271 A_24_P250922   PTGS2
##  ---                                
##  97: -1.8709626   A_23_P1691    MMP1
##  98: -3.1170994  A_23_P79518    IL1B
##  99: -1.1331159 A_23_P374844     GAL
## 100: -2.2965098 A_23_P122924   INHBA
## 101: -1.9277140   A_23_P1691    MMP1

with scaling by row

Heatmap with probe means

with scaling by row

GO:0001755 Human genes

##  [1] "GBX2"    "KITLG"   "SEMA7A"  "LAMA5"   "SEMA6A"  "TBX1"    "SHH"    
##  [8] "GDNF"    "HIF1A"   "SEMA4D"  "HTR2B"   "CORO1C"  "EFNB1"   "ERBB4"  
## [15] "LEF1"    "NRTN"    "FGF19"   "SEMA6D"  "SEMA5B"  "RIC8A"   "SEMA3A" 
## [22] "EDN3"    "SEMA5A"  "SOX8"    "SEMA3G"  "SEMA4A"  "ISL1"    "SEMA3B" 
## [29] "SMO"     "ACVR1"   "SEMA4G"  "SEMA6B"  "SOX10"   "EDNRB"   "SEMA4F" 
## [36] "SEMA3F"  "ALX1"    "SEMA6C"  "OVOL2"   "SEMA4C"  "PHACTR4" "SEMA3E" 
## [43] "NRP2"    "SEMA3D"  "SEMA3C"  "RET"