qPCR: young and old Legumain knock out mice compared to their Wild type and Heterozygous counterparts
TS
sep 2021
1 Background
Kidney, Liver, Lung and Spleen samples from young and old Legumain knock out, wild type and old heterozygots mice were probed for relative gene expression of CHIA, CHIL3, EPX, MPO and SOD1 with qPCR (SYBR).
Above is a figure presenting relative expression of CHIA, EPX, MPO and SOD1 from Human protein atlas. Organs of interest are marked with blue square. Based on this info, we can expect CHIA to be expreseed in lungs, EPX in spleen and lungs, MPO plausibly in spleen and lungs and for SOD1 to appear in all probed organs
The quality of results is mostly in accordance with above listed expectations. Table below indicates which experiments are valid (✓) and which are not (X). Latter is due to no signal (No Ct), too high Ct values or too wide dispersion among technical replicates, all of which indicates to low aboundance of target gene in the sample. To clarify, only results with ✓ should be considerd further.
| Gene/ Tissue | Kidney | Liver | Lung | Spleen |
|---|---|---|---|---|
| CHIA | X | X | ✓ | X |
| CHIL3 | ✓ | X | ✓ | ✓ |
| EPX | X | X | X | ✓ |
| MPO | X | X | X | ✓ |
| SOD1 | ✓ | ✓ | ✓ | ✓ |
How to read: KO -> Knock out
WT -> Wild type
H -> Heterozygotes
ML -> Mladi
ST -> Stari
Exsample: KO_ML -> knock outs, mladi
Plots: different letter (and colour) indicate statistically significant difference between samples (eg.: sample marked with “a” is different from sample marked “b”, but it is not different from sample marked “ab”). \(\alpha\) (level of significance) is set to 0.05. On horizontal axis there are samples (different genotype and age of the mice) while on vertical axis there is fold difference in expression. In this file, fold differences are not logarithmised.
Note, expression is calculated relevant to WT_ML (young wild types). Note, for the ease with work, ‘No Ct’ values in raw data have been replaced with “40” in this file. So, where there was no target gene detected whatosoever, data has been replaced so that it indicates very low amount of that target in the sample.
Link to .Rmd output without {r include=FALSE} : here
2 Kidney
Urejanje podatkov povprecimo posamezne skupine, normalizacija na standard (B2M)
\(\Delta\)Ct = Ct (gene of interest ) - Ct (housekeeping gene)
\(\Delta\Delta\)Ct = \(\Delta\)Ct (sample) - \(\Delta\)Ct (control average) control average je WM -> ekspresijo zracunamo relativno na WM
fold gene gxpression relative to WM 2^-(\(\Delta\Delta\)Ct)
ANOVA
2.0.1 CHIA
results OK? : X
preverjamo kombiniran vpliv starosti in genotipa
razlika med posameznimi vzorci, vse je glede na WM
2.0.1.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = CHIA ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -0.50345052 -5.076708 4.069807 0.9956956
## KO_ST-H 9.76943789 5.196180 14.342696 0.0002683
## WT_ML-H -0.40761946 -4.980877 4.165638 0.9980961
## WT_ST-H 0.39161452 -4.181643 4.964872 0.9983711
## KO_ST-KO_ML 10.27288841 5.699631 14.846146 0.0001759
## WT_ML-KO_ML 0.09583107 -4.477427 4.669089 0.9999939
## WT_ST-KO_ML 0.89506504 -3.678193 5.468323 0.9639090
## WT_ML-KO_ST -10.17705735 -14.750315 -5.603800 0.0001904
## WT_ST-KO_ST -9.37782337 -13.951081 -4.804566 0.0003763
## WT_ST-WT_ML 0.79923398 -3.774024 5.372492 0.9758572##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for CHIA
##
## Mean Square Error: 2.896444
##
## AG, means
##
## CHIA std r Min Max
## H 1.4121559 0.1481574 3 1.3088805 1.581909
## KO_ML 0.9087053 0.4903553 3 0.5690655 1.470867
## KO_ST 11.1815938 3.6714344 3 7.1519336 14.336954
## WT_ML 1.0045364 0.1156086 3 0.8806659 1.109569
## WT_ST 1.8037704 0.8526582 3 1.0081195 2.703822
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 3.096201 3.235502 3.317499 3.369951
##
## Means with the same letter are not significantly different.
##
## CHIA groups
## KO_ST 11.1815938 a
## WT_ST 1.8037704 b
## H 1.4121559 b
## WT_ML 1.0045364 b
## KO_ML 0.9087053 b
2.0.2 CHIL3
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
2.0.2.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = CHIL3 ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -2.9971219 -19.124816 13.130573 0.9699321
## KO_ST-H 17.8996124 1.771918 34.027307 0.0285265
## WT_ML-H -2.3710719 -18.498766 13.756623 0.9871656
## WT_ST-H -1.4717764 -17.599471 14.655918 0.9979134
## KO_ST-KO_ML 20.8967344 4.769040 37.024429 0.0111870
## WT_ML-KO_ML 0.6260500 -15.501644 16.753744 0.9999283
## WT_ST-KO_ML 1.5253456 -14.602349 17.653040 0.9976029
## WT_ML-KO_ST -20.2706843 -36.398379 -4.142990 0.0135721
## WT_ST-KO_ST -19.3713888 -35.499083 -3.243694 0.0179567
## WT_ST-WT_ML 0.8992955 -15.228399 17.026990 0.9996983##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for CHIL3
##
## Mean Square Error: 36.02122
##
## AG, means
##
## CHIL3 std r Min Max
## H 3.719959 0.4704884 3 3.1882788 4.082476
## KO_ML 0.722837 0.2712621 3 0.5655701 1.036063
## KO_ST 21.619571 13.3254172 3 9.1436981 35.656943
## WT_ML 1.348887 1.3119746 3 0.5642649 2.863496
## WT_ST 2.248183 0.7232694 3 1.7647263 3.079674
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 10.91882 11.41007 11.69923 11.88421
##
## Means with the same letter are not significantly different.
##
## CHIL3 groups
## KO_ST 21.619571 a
## H 3.719959 b
## WT_ST 2.248183 b
## WT_ML 1.348887 b
## KO_ML 0.722837 b
2.0.3 EPX
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM
2.0.3.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = EPX ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -2.7920170 -28.98006 23.39603 0.9961938
## KO_ST-H 11.6643712 -14.52367 37.85241 0.6038388
## WT_ML-H -2.0280269 -28.21607 24.16002 0.9989002
## WT_ST-H -2.2002505 -28.38829 23.98779 0.9984876
## KO_ST-KO_ML 14.4563882 -11.73166 40.64443 0.4155995
## WT_ML-KO_ML 0.7639901 -25.42405 26.95203 0.9999770
## WT_ST-KO_ML 0.5917665 -25.59628 26.77981 0.9999917
## WT_ML-KO_ST -13.6923981 -39.88044 12.49565 0.4641835
## WT_ST-KO_ST -13.8646217 -40.05267 12.32342 0.4529857
## WT_ST-WT_ML -0.1722236 -26.36027 26.01582 0.9999999##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for EPX
##
## Mean Square Error: 94.97732
##
## AG, means
##
## EPX std r Min Max
## H 3.953632 0.4729797 3 3.4461846 4.382234
## KO_ML 1.161615 0.4777912 3 0.8745827 1.713168
## KO_ST 15.618004 21.6276813 3 1.9207459 40.551030
## WT_ML 1.925605 2.5308258 3 0.3634931 4.845571
## WT_ST 1.753382 0.5224203 3 1.4028233 2.353813
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 17.72991 18.52759 18.99714 19.29750
##
## Means with the same letter are not significantly different.
##
## EPX groups
## KO_ST 15.618004 a
## H 3.953632 a
## WT_ML 1.925605 a
## WT_ST 1.753382 a
## KO_ML 1.161615 a
2.0.4 MPO
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM podatki niso pravi, MPO se ni izrazal
## Df Sum Sq Mean Sq F value Pr(>F)
## AG 4 520.3 130.07 3.909 0.0366 *
## Residuals 10 332.7 33.27
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 12.0.4.1 post-hoc
2.0.5 SOD1
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
2.0.5.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = SOD1 ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -2.04380150 -4.546931 0.4593277 0.1261792
## KO_ST-H -0.99831771 -3.501447 1.5048115 0.6903003
## WT_ML-H -1.33223075 -3.835360 1.1708984 0.4482590
## WT_ST-H -1.32171164 -3.824841 1.1814175 0.4553614
## KO_ST-KO_ML 1.04548379 -1.457645 3.5486130 0.6554885
## WT_ML-KO_ML 0.71157075 -1.791558 3.2146999 0.8767173
## WT_ST-KO_ML 0.72208985 -1.781039 3.2252190 0.8711221
## WT_ML-KO_ST -0.33391304 -2.837042 2.1692161 0.9910728
## WT_ST-KO_ST -0.32339394 -2.826523 2.1797352 0.9920864
## WT_ST-WT_ML 0.01051911 -2.492610 2.5136483 1.0000000##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for SOD1
##
## Mean Square Error: 0.8677214
##
## AG, means
##
## SOD1 std r Min Max
## H 2.5218236 1.5052781 3 0.7836782 3.3908963
## KO_ML 0.4780221 0.2656565 3 0.2126668 0.7439788
## KO_ST 1.5235059 1.0924641 3 0.6484198 2.7479073
## WT_ML 1.1895929 0.8704238 3 0.5396141 2.1784973
## WT_ST 1.2001120 0.2259567 3 0.9850940 1.4356128
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 1.694676 1.770921 1.815801 1.844511
##
## Means with the same letter are not significantly different.
##
## SOD1 groups
## H 2.5218236 a
## KO_ST 1.5235059 ab
## WT_ST 1.2001120 ab
## WT_ML 1.1895929 ab
## KO_ML 0.4780221 b
3 Liver
Urejanje podatkov povprecimo posamezne skupine, normalizacija na standard (B2M)
\(\Delta\)Ct = Ct (gene of interest ) - Ct (housekeeping gene)
\(\Delta\Delta\)Ct = \(\Delta\)Ct (sample) - \(\Delta\)Ct (control average) control average je WM, ekspresijo zracunamo relativno na WM
fold gene gxpression relative to WM 2^-(\(\Delta\Delta\)Ct)
##ANOVA
3.0.1 CHIA
#preverjamo kombiniran vpliv starosti in genotipa
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM
3.0.1.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = CHIA ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 1.6515145 -2.752235 6.055264 0.7333737
## KO_ST-H 9.4634191 5.059670 13.867168 0.0002553
## WT_ML-H 0.4947731 -3.908976 4.898522 0.9953462
## WT_ST-H 1.9287735 -2.474976 6.332523 0.6176639
## KO_ST-KO_ML 7.8119046 3.408156 12.215654 0.0011968
## WT_ML-KO_ML -1.1567413 -5.560490 3.247008 0.9034916
## WT_ST-KO_ML 0.2772590 -4.126490 4.681008 0.9995126
## WT_ML-KO_ST -8.9686460 -13.372395 -4.564897 0.0003980
## WT_ST-KO_ST -7.5346456 -11.938395 -3.130897 0.0015794
## WT_ST-WT_ML 1.4340003 -2.969749 5.837749 0.8167033##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for CHIA
##
## Mean Square Error: 2.685709
##
## AG, means
##
## CHIA std r Min Max
## H 0.5097633 0.05348221 3 0.4724827 0.5710412
## KO_ML 2.1612777 1.16626808 3 1.3534735 3.4983307
## KO_ST 9.9731824 3.27465706 3 6.3790135 12.7875384
## WT_ML 1.0045364 0.11560862 3 0.8806659 1.1095695
## WT_ST 2.4385367 1.15271788 3 1.3628877 3.6553258
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 2.981440 3.115577 3.194535 3.245043
##
## Means with the same letter are not significantly different.
##
## CHIA groups
## KO_ST 9.9731824 a
## WT_ST 2.4385367 b
## KO_ML 2.1612777 b
## WT_ML 1.0045364 b
## H 0.5097633 b
3.0.2 CHIL3
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM
3.0.2.1 post-hoc
3.0.3 EPX
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM
3.0.3.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = EPX ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 1.3356112 -22.10102 24.77224 0.9996712
## KO_ST-H 12.5029515 -10.93368 35.93958 0.4461525
## WT_ML-H 0.4984141 -22.93822 23.93504 0.9999935
## WT_ST-H 0.9432247 -22.49340 24.37985 0.9999173
## KO_ST-KO_ML 11.1673403 -12.26929 34.60397 0.5464475
## WT_ML-KO_ML -0.8371971 -24.27383 22.59943 0.9999485
## WT_ST-KO_ML -0.3923866 -23.82902 23.04424 0.9999975
## WT_ML-KO_ST -12.0045374 -35.44117 11.43209 0.4825715
## WT_ST-KO_ST -11.5597268 -34.99636 11.87690 0.5161470
## WT_ST-WT_ML 0.4448105 -22.99182 23.88144 0.9999959##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for EPX
##
## Mean Square Error: 76.06837
##
## AG, means
##
## EPX std r Min Max
## H 1.427191 0.1707373 3 1.2440117 1.581909
## KO_ML 2.762803 1.1363855 3 2.0801199 4.074623
## KO_ST 13.930143 19.2903458 3 1.7131680 36.168620
## WT_ML 1.925605 2.5308258 3 0.3634931 4.845571
## WT_ST 2.370416 0.7062657 3 1.8964921 3.182146
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 15.86714 16.58102 17.00123 17.27003
##
## Means with the same letter are not significantly different.
##
## EPX groups
## KO_ST 13.930143 a
## KO_ML 2.762803 a
## WT_ST 2.370416 a
## WT_ML 1.925605 a
## H 1.427191 a
3.0.4 MPO
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM
3.0.4.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = MPO ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 12.88636302 -14.42497 40.19769 0.5549712
## KO_ST-H 14.46716456 -12.84417 41.77849 0.4524997
## WT_ML-H 0.09572773 -27.21560 27.40706 1.0000000
## WT_ST-H 2.92109274 -24.39024 30.23242 0.9961468
## KO_ST-KO_ML 1.58080154 -25.73053 28.89213 0.9996504
## WT_ML-KO_ML -12.79063529 -40.10197 14.52069 0.5614064
## WT_ST-KO_ML -9.96527029 -37.27660 17.34606 0.7512756
## WT_ML-KO_ST -14.37143683 -41.68277 12.93989 0.4584478
## WT_ST-KO_ST -11.54607183 -38.85740 15.76526 0.6460307
## WT_ST-WT_ML 2.82536500 -24.48596 30.13669 0.9966104##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for MPO
##
## Mean Square Error: 103.2998
##
## AG, means
##
## MPO std r Min Max
## H 0.9042723 0.5679899 3 0.5763432 1.560130
## KO_ML 13.7906353 21.0132338 3 1.6586391 38.054628
## KO_ST 15.3714368 7.9629172 3 6.8210791 22.575197
## WT_ML 1.0000000 0.0000000 3 1.0000000 1.000000
## WT_ST 3.8253650 3.3484867 3 1.8921153 7.691864
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 18.49040 19.32230 19.81198 20.12522
##
## Means with the same letter are not significantly different.
##
## MPO groups
## KO_ST 15.3714368 a
## KO_ML 13.7906353 a
## WT_ST 3.8253650 a
## WT_ML 1.0000000 a
## H 0.9042723 a
3.0.5 SOD1
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
3.0.5.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = SOD1 ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 0.22660092 -1.671564 2.124765 0.9941341
## KO_ST-H 0.44852468 -1.449640 2.346689 0.9314387
## WT_ML-H 0.27925918 -1.618905 2.177424 0.9871326
## WT_ST-H 0.71211069 -1.186054 2.610275 0.7331371
## KO_ST-KO_ML 0.22192376 -1.676241 2.120088 0.9945820
## WT_ML-KO_ML 0.05265826 -1.845506 1.950823 0.9999812
## WT_ST-KO_ML 0.48550977 -1.412655 2.383674 0.9113101
## WT_ML-KO_ST -0.16926550 -2.067430 1.728899 0.9980926
## WT_ST-KO_ST 0.26358601 -1.634578 2.161750 0.9896227
## WT_ST-WT_ML 0.43285151 -1.465313 2.331016 0.9390785##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for SOD1
##
## Mean Square Error: 0.4989781
##
## AG, means
##
## SOD1 std r Min Max
## H 0.9103337 0.5433787 3 0.2828940 1.224054
## KO_ML 1.1369346 0.6318412 3 0.5058097 1.769490
## KO_ST 1.3588584 0.9743999 3 0.5783441 2.450937
## WT_ML 1.1895929 0.8704238 3 0.5396141 2.178497
## WT_ST 1.6224444 0.3054734 3 1.3317593 1.940820
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 1.285101 1.342919 1.376952 1.398723
##
## Means with the same letter are not significantly different.
##
## SOD1 groups
## WT_ST 1.6224444 a
## KO_ST 1.3588584 a
## WT_ML 1.1895929 a
## KO_ML 1.1369346 a
## H 0.9103337 a
4 Lung
Urejanje podatkov povprecimo posamezne skupine, normalizacija na standard (B2M)
\(\Delta\)Ct = Ct (gene of interest ) - Ct (housekeeping gene)
\(\Delta\Delta\)Ct = \(\Delta\)Ct (sample) - \(\Delta\)Ct (control average) control average je WM, ekspresijo zracunamo relativno na WM
fold gene gxpression relative to WM 2^-(\(\Delta\Delta\)Ct)
##ANOVA
4.0.1 CHIA
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
4.0.1.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = CHIA ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -0.5374947 -2.7973351 1.7223457 0.9299429
## KO_ST-H 1.2786303 -0.9812101 3.5384707 0.3936884
## WT_ML-H -0.1485465 -2.4083869 2.1112939 0.9994224
## WT_ST-H 0.3706934 -1.8891470 2.6305338 0.9808035
## KO_ST-KO_ML 1.8161249 -0.4437155 4.0759653 0.1344059
## WT_ML-KO_ML 0.3889482 -1.8708922 2.6487886 0.9771496
## WT_ST-KO_ML 0.9081881 -1.3516523 3.1680285 0.6846908
## WT_ML-KO_ST -1.4271768 -3.6870171 0.8326636 0.2995282
## WT_ST-KO_ST -0.9079368 -3.1677772 1.3519036 0.6848953
## WT_ST-WT_ML 0.5192399 -1.7406005 2.7790803 0.9375205##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for CHIA
##
## Mean Square Error: 0.7072441
##
## AG, means
##
## CHIA std r Min Max
## H 1.1810261 1.0240715 3 0.04503599 2.0333927
## KO_ML 0.6435314 0.1781955 3 0.44203185 0.7803657
## KO_ST 2.4596564 0.8149452 3 1.51980780 2.9701907
## WT_ML 1.0324796 0.3266307 3 0.76607394 1.3968937
## WT_ST 1.5517195 1.2980452 3 0.36195667 2.9360751
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 1.529964 1.598799 1.639317 1.665235
##
## Means with the same letter are not significantly different.
##
## CHIA groups
## KO_ST 2.4596564 a
## WT_ST 1.5517195 ab
## H 1.1810261 ab
## WT_ML 1.0324796 ab
## KO_ML 0.6435314 b
4.0.2 CHIL3
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
4.0.2.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = CHIL3 ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 0.05149904 -3.415266 3.518264 0.9999984
## KO_ST-H 0.08085619 -3.385909 3.547621 0.9999906
## WT_ML-H 0.73920027 -2.727565 4.205965 0.9514530
## WT_ST-H 2.24416270 -1.222602 5.710928 0.2795606
## KO_ST-KO_ML 0.02935716 -3.437408 3.496122 0.9999998
## WT_ML-KO_ML 0.68770124 -2.779064 4.154466 0.9621701
## WT_ST-KO_ML 2.19266367 -1.274101 5.659429 0.2983077
## WT_ML-KO_ST 0.65834408 -2.808421 4.125109 0.9675401
## WT_ST-KO_ST 2.16330651 -1.303459 5.630072 0.3094073
## WT_ST-WT_ML 1.50496243 -1.961803 4.971727 0.6248814##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for CHIL3
##
## Mean Square Error: 1.664419
##
## AG, means
##
## CHIL3 std r Min Max
## H 0.2633804 0.22095891 3 0.01098491 0.4219079
## KO_ML 0.3148795 0.06728670 3 0.27043117 0.3922920
## KO_ST 0.3442366 0.11300848 3 0.21638414 0.4307731
## WT_ML 1.0025807 0.08769858 3 0.91172249 1.0867349
## WT_ST 2.5075431 2.87198221 3 0.58709532 5.8091752
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 2.347080 2.452677 2.514835 2.554596
##
## Means with the same letter are not significantly different.
##
## CHIL3 groups
## WT_ST 2.5075431 a
## WT_ML 1.0025807 a
## KO_ST 0.3442366 a
## KO_ML 0.3148795 a
## H 0.2633804 a
4.0.3 EPX
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM
4.0.3.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = EPX ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 1.492970 -38.88643 41.872371 0.9999410
## KO_ST-H 28.953068 -11.42633 69.332470 0.2036595
## WT_ML-H -5.678702 -46.05810 34.700700 0.9891200
## WT_ST-H -3.872552 -44.25195 36.506850 0.9974701
## KO_ST-KO_ML 27.460098 -12.91930 67.839500 0.2414979
## WT_ML-KO_ML -7.171671 -47.55107 33.207730 0.9744164
## WT_ST-KO_ML -5.365521 -45.74492 35.013880 0.9912030
## WT_ML-KO_ST -34.631770 -75.01117 5.747632 0.1028571
## WT_ST-KO_ST -32.825620 -73.20502 7.553782 0.1284169
## WT_ST-WT_ML 1.806150 -38.57325 42.185552 0.9998741##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for EPX
##
## Mean Square Error: 225.805
##
## AG, means
##
## EPX std r Min Max
## H 7.384493 6.399709 3 3.0915564 14.740021
## KO_ML 8.877463 8.182013 3 3.1748021 18.252219
## KO_ST 36.337561 31.699586 3 6.2984638 69.470729
## WT_ML 1.705792 2.100952 3 0.4622248 4.131503
## WT_ST 3.511942 3.441754 3 0.7569833 7.370010
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 27.33779 28.56774 29.29173 29.75485
##
## Means with the same letter are not significantly different.
##
## EPX groups
## KO_ST 36.337561 a
## KO_ML 8.877463 b
## H 7.384493 b
## WT_ST 3.511942 b
## WT_ML 1.705792 b
4.0.4 MPO
results OK? : X
razlika med posameznimi vzorci, vse je glede na WM
4.0.4.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = MPO ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 4.9498060 -2.2992970 12.1989090 0.2384822
## KO_ST-H 8.1221891 0.8730861 15.3712921 0.0270309
## WT_ML-H 0.5524596 -6.6966435 7.8015626 0.9989671
## WT_ST-H 0.3457424 -6.9033607 7.5948454 0.9998376
## KO_ST-KO_ML 3.1723831 -4.0767199 10.4214861 0.6183351
## WT_ML-KO_ML -4.3973464 -11.6464495 2.8517566 0.3331631
## WT_ST-KO_ML -4.6040636 -11.8531667 2.6450394 0.2949079
## WT_ML-KO_ST -7.5697295 -14.8188326 -0.3206265 0.0398835
## WT_ST-KO_ST -7.7764467 -15.0255498 -0.5273437 0.0344754
## WT_ST-WT_ML -0.2067172 -7.4558202 7.0423858 0.9999790##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for MPO
##
## Mean Square Error: 7.277502
##
## AG, means
##
## MPO std r Min Max
## H 0.5581983 0.3598352 3 0.2001904 0.9198334
## KO_ML 5.5080043 4.0233691 3 1.9603498 9.8795439
## KO_ST 8.6803874 4.4099831 3 4.5507714 13.3254386
## WT_ML 1.1106579 0.5305322 3 0.4980783 1.4218584
## WT_ST 0.9039407 0.5840507 3 0.2551548 1.3877789
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 4.907811 5.128617 5.258591 5.341733
##
## Means with the same letter are not significantly different.
##
## MPO groups
## KO_ST 8.6803874 a
## KO_ML 5.5080043 ab
## WT_ML 1.1106579 b
## WT_ST 0.9039407 b
## H 0.5581983 b
4.0.5 SOD1
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
4.0.5.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = SOD1 ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 0.24889807 -0.6627233 1.1605194 0.8910781
## KO_ST-H 1.48818658 0.5765652 2.3998079 0.0022484
## WT_ML-H -0.03297658 -0.9445979 0.8786448 0.9999459
## WT_ST-H -0.39199403 -1.3036154 0.5196273 0.6325498
## KO_ST-KO_ML 1.23928851 0.3276672 2.1509098 0.0081725
## WT_ML-KO_ML -0.28187464 -1.1934960 0.6297467 0.8418091
## WT_ST-KO_ML -0.64089210 -1.5525134 0.2707292 0.2173601
## WT_ML-KO_ST -1.52116315 -2.4327845 -0.6095418 0.0019088
## WT_ST-KO_ST -1.88018061 -2.7918019 -0.9685593 0.0003588
## WT_ST-WT_ML -0.35901745 -1.2706388 0.5526039 0.6994595##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for SOD1
##
## Mean Square Error: 0.1150914
##
## AG, means
##
## SOD1 std r Min Max
## H 1.0394439 0.3105703 3 0.71449707 1.333299
## KO_ML 1.2883420 0.2629440 3 1.04246576 1.565547
## KO_ST 2.5276305 0.3377030 3 2.25271791 2.904588
## WT_ML 1.0064673 0.1403753 3 0.87458267 1.154019
## WT_ST 0.6474499 0.5254663 3 0.09034989 1.134193
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 0.6171887 0.6449566 0.6613016 0.6717573
##
## Means with the same letter are not significantly different.
##
## SOD1 groups
## KO_ST 2.5276305 a
## KO_ML 1.2883420 b
## H 1.0394439 b
## WT_ML 1.0064673 b
## WT_ST 0.6474499 b
5 Spleen
Urejanje podatkov povprecimo posamezne skupine, normalizacija na standard (B2M)
\(\Delta\)Ct = Ct (gene of interest ) - Ct (housekeeping gene)
\(\Delta\Delta\)Ct = \(\Delta\)Ct (sample) - \(\Delta\)Ct (control average) control average je WM, ekspresijo zracunamo relativno na WM
fold gene gxpression relative to WM 2^-(\(\Delta\Delta\)Ct)
##ANOVA
5.0.1 CHIA
results OK? : X
preverjamo kombiniran vpliv starosti in genotipa
razlika med posameznimi vzorci, vse je glede na WM
5.0.1.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = CHIA ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -0.3237812 -6.313412 5.665850 0.9997332
## KO_ST-H 2.7309953 -3.258636 8.720626 0.5842517
## WT_ML-H 0.2759131 -5.713718 6.265544 0.9998585
## WT_ST-H -0.1284295 -6.118060 5.861201 0.9999933
## KO_ST-KO_ML 3.0547765 -2.934854 9.044407 0.4864184
## WT_ML-KO_ML 0.5996943 -5.389936 6.589325 0.9970120
## WT_ST-KO_ML 0.1953517 -5.794279 6.184983 0.9999642
## WT_ML-KO_ST -2.4550822 -8.444713 3.534549 0.6699170
## WT_ST-KO_ST -2.8594248 -8.849056 3.130206 0.5447960
## WT_ST-WT_ML -0.4043426 -6.393973 5.585288 0.9993585##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for CHIA
##
## Mean Square Error: 4.96837
##
## AG, means
##
## CHIA std r Min Max
## H 0.8311363 0.4716140 3 0.2892834 1.1491408
## KO_ML 0.5073551 0.2959930 3 0.1678862 0.7114769
## KO_ST 3.5621316 4.9145259 3 0.6229647 9.2357058
## WT_ML 1.1070494 0.5226064 3 0.5042539 1.4328513
## WT_ST 0.7027068 0.3257830 3 0.4476849 1.0697118
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 4.055119 4.237562 4.344954 4.413651
##
## Means with the same letter are not significantly different.
##
## CHIA groups
## KO_ST 3.5621316 a
## WT_ML 1.1070494 a
## H 0.8311363 a
## WT_ST 0.7027068 a
## KO_ML 0.5073551 a
5.0.2 CHIL3
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
5.0.2.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = CHIL3 ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 0.3427559 -6.810550 7.496062 0.9998346
## KO_ST-H 1.0407560 -6.112550 8.194062 0.9876530
## WT_ML-H -0.8232574 -7.976564 6.330049 0.9948981
## WT_ST-H 2.0048417 -5.148464 9.158148 0.8819349
## KO_ST-KO_ML 0.6980001 -6.455306 7.851306 0.9972950
## WT_ML-KO_ML -1.1660133 -8.319319 5.987293 0.9812398
## WT_ST-KO_ML 1.6620858 -5.491220 8.815392 0.9351522
## WT_ML-KO_ST -1.8640134 -9.017320 5.289293 0.9059015
## WT_ST-KO_ST 0.9640857 -6.189220 8.117392 0.9907218
## WT_ST-WT_ML 2.8280991 -4.325207 9.981405 0.6966598##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for CHIL3
##
## Mean Square Error: 7.086429
##
## AG, means
##
## CHIL3 std r Min Max
## H 1.932440 1.019509 3 1.2948435 3.108268
## KO_ML 2.275195 1.740457 3 0.2873958 3.525378
## KO_ST 2.973196 2.113024 3 1.6850341 5.411810
## WT_ML 1.109182 0.528815 3 0.5015427 1.465214
## WT_ST 3.937281 5.159364 3 0.7752738 9.890964
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 4.842954 5.060842 5.189098 5.271142
##
## Means with the same letter are not significantly different.
##
## CHIL3 groups
## WT_ST 3.937281 a
## KO_ST 2.973196 a
## KO_ML 2.275195 a
## H 1.932440 a
## WT_ML 1.109182 a
5.0.3 EPX
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
5.0.3.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = EPX ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H 0.5254257 -2.913931 3.964782 0.9852095
## KO_ST-H 1.3229386 -2.116418 4.762295 0.7161208
## WT_ML-H 0.1869128 -3.252444 3.626269 0.9997275
## WT_ST-H 0.7364552 -2.702901 4.175812 0.9507488
## KO_ST-KO_ML 0.7975130 -2.641844 4.236870 0.9355902
## WT_ML-KO_ML -0.3385129 -3.777870 3.100844 0.9972032
## WT_ST-KO_ML 0.2110295 -3.228327 3.650386 0.9995598
## WT_ML-KO_ST -1.1360259 -4.575383 2.303331 0.8092943
## WT_ST-KO_ST -0.5864834 -4.025840 2.852873 0.9779036
## WT_ST-WT_ML 0.5495425 -2.889814 3.988899 0.9825593##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for EPX
##
## Mean Square Error: 1.638205
##
## AG, means
##
## EPX std r Min Max
## H 0.8985375 0.7167724 3 0.3287357 1.703301
## KO_ML 1.4239632 1.1570281 3 0.2426026 2.555018
## KO_ST 2.2214761 1.1583603 3 1.3456781 3.534893
## WT_ML 1.0854503 0.4968070 3 0.5677522 1.558329
## WT_ST 1.6349927 2.1794340 3 0.2324512 4.145846
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 2.328524 2.433286 2.494953 2.534400
##
## Means with the same letter are not significantly different.
##
## EPX groups
## KO_ST 2.2214761 a
## WT_ST 1.6349927 a
## KO_ML 1.4239632 a
## WT_ML 1.0854503 a
## H 0.8985375 a
5.0.4 MPO
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
5.0.4.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = MPO ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -2.732215 -47.72483 42.26040 0.9995774
## KO_ST-H 2.626416 -42.36620 47.61903 0.9996384
## WT_ML-H -9.190419 -54.18303 35.80219 0.9581171
## WT_ST-H 11.664735 -33.32788 56.65735 0.9074076
## KO_ST-KO_ML 5.358630 -39.63398 50.35124 0.9941861
## WT_ML-KO_ML -6.458204 -51.45082 38.53441 0.9882583
## WT_ST-KO_ML 14.396950 -30.59566 59.38956 0.8255053
## WT_ML-KO_ST -11.816834 -56.80945 33.17578 0.9035294
## WT_ST-KO_ST 9.038319 -35.95429 54.03093 0.9604731
## WT_ST-WT_ML 20.855154 -24.13746 65.84777 0.5702528##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for MPO
##
## Mean Square Error: 280.3472
##
## AG, means
##
## MPO std r Min Max
## H 10.306124 5.4078933 3 5.7734931 16.29222
## KO_ML 7.573909 10.7298239 3 0.7637176 19.94255
## KO_ST 12.932539 14.5994458 3 2.2648954 29.57100
## WT_ML 1.115705 0.5475276 3 0.4934963 1.52391
## WT_ST 21.970859 32.3097194 3 3.2402634 59.27880
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 30.46104 31.83151 32.63821 33.15424
##
## Means with the same letter are not significantly different.
##
## MPO groups
## WT_ST 21.970859 a
## KO_ST 12.932539 a
## H 10.306124 a
## KO_ML 7.573909 a
## WT_ML 1.115705 a
5.0.5 SOD1
results OK? : ✓
razlika med posameznimi vzorci, vse je glede na WM
5.0.5.1 post-hoc
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = SOD1 ~ AG, data = lg)
##
## $AG
## diff lwr upr p adj
## KO_ML-H -0.419652049 -1.7973871 0.9580830 0.8485607
## KO_ST-H 0.008762409 -1.3689726 1.3864975 0.9999999
## WT_ML-H -0.424642374 -1.8023774 0.9530927 0.8432621
## WT_ST-H -0.246697272 -1.6244323 1.1310378 0.9736592
## KO_ST-KO_ML 0.428414458 -0.9493206 1.8061495 0.8392036
## WT_ML-KO_ML -0.004990326 -1.3827254 1.3727447 1.0000000
## WT_ST-KO_ML 0.172954777 -1.2047803 1.5506898 0.9929008
## WT_ML-KO_ST -0.433404784 -1.8111398 0.9443303 0.8337655
## WT_ST-KO_ST -0.255459681 -1.6331947 1.1222754 0.9701708
## WT_ST-WT_ML 0.177945103 -1.1997899 1.5556802 0.9920952##
## Study: test ~ "AG"
##
## Duncan's new multiple range test
## for SOD1
##
## Mean Square Error: 0.2628725
##
## AG, means
##
## SOD1 std r Min Max
## H 1.429875 0.4888104 3 0.9177106 1.891387
## KO_ML 1.010223 0.6977118 3 0.4083216 1.774949
## KO_ST 1.438637 0.6130253 3 0.9544740 2.127921
## WT_ML 1.005233 0.1235268 3 0.8692107 1.110424
## WT_ST 1.183178 0.4444843 3 0.6890964 1.550548
##
## Alpha: 0.05 ; DF Error: 10
##
## Critical Range
## 2 3 4 5
## 0.9327585 0.9747241 0.9994263 1.0152280
##
## Means with the same letter are not significantly different.
##
## SOD1 groups
## KO_ST 1.438637 a
## H 1.429875 a
## WT_ST 1.183178 a
## KO_ML 1.010223 a
## WT_ML 1.005233 a
6 Combined box plots
Data in these plots was normalised with log10()
## R version 4.0.5 (2021-03-31)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## Running under: Windows 10 x64 (build 19042)
##
## Matrix products: default
##
## locale:
## [1] LC_COLLATE=English_United Kingdom.1252
## [2] LC_CTYPE=English_United Kingdom.1252
## [3] LC_MONETARY=English_United Kingdom.1252
## [4] LC_NUMERIC=C
## [5] LC_TIME=English_United Kingdom.1252
## system code page: 1250
##
## attached base packages:
## [1] grid stats graphics grDevices utils datasets methods
## [8] base
##
## other attached packages:
## [1] writexl_1.4.0 data.table_1.12.8 plyr_1.8.6
## [4] AICcmodavg_2.3-1 readr_2.0.1 DescTools_0.99.42
## [7] gplots_3.1.1 RColorBrewer_1.1-2 ape_5.5
## [10] ggdendro_0.1.22 ggplot2_3.3.5 mvnormtest_0.1-9
## [13] readxl_1.3.1 lawstat_3.4 HH_3.1-43
## [16] gridExtra_2.3 multcomp_1.4-17 TH.data_1.0-10
## [19] MASS_7.3-53.1 survival_3.2-10 mvtnorm_1.1-2
## [22] latticeExtra_0.6-29 Rmpfr_0.8-4 gmp_0.6-2
## [25] lattice_0.20-44 agricolae_1.3-5
##
## loaded via a namespace (and not attached):
## [1] VGAM_1.1-5 colorspace_1.4-1 class_7.3-18
## [4] ellipsis_0.3.2 htmlTable_2.2.1 base64enc_0.1-3
## [7] gld_2.6.2 proxy_0.4-26 rstudioapi_0.13
## [10] farver_2.1.0 fansi_0.4.1 codetools_0.2-18
## [13] splines_4.0.5 leaps_3.1 rootSolve_1.8.2.2
## [16] knitr_1.33 Formula_1.2-4 jsonlite_1.7.2
## [19] cluster_2.1.1 png_0.1-7 Kendall_2.2
## [22] shiny_1.6.0 compiler_4.0.5 backports_1.2.1
## [25] assertthat_0.2.1 Matrix_1.3-2 fastmap_1.1.0
## [28] later_1.3.0 htmltools_0.5.2 tools_4.0.5
## [31] lmom_2.8 gtable_0.3.0 glue_1.4.1
## [34] reshape2_1.4.4 dplyr_1.0.6 Rcpp_1.0.7
## [37] raster_3.4-13 cellranger_1.1.0 jquerylib_0.1.4
## [40] vctrs_0.3.8 nlme_3.1-152 lmtest_0.9-38
## [43] xfun_0.22 stringr_1.4.0 rbibutils_2.2.3
## [46] mime_0.11 miniUI_0.1.1.1 lifecycle_1.0.0
## [49] gtools_3.9.2 zoo_1.8-9 scales_1.1.1
## [52] hms_1.1.0 promises_1.2.0.1 parallel_4.0.5
## [55] sandwich_3.0-1 expm_0.999-6 Exact_2.1
## [58] yaml_2.2.1 sass_0.4.0 labelled_2.8.0
## [61] rpart_4.1-15 stringi_1.5.3 highr_0.9
## [64] klaR_0.6-15 AlgDesign_1.2.0 e1071_1.7-8
## [67] checkmate_2.0.0 caTools_1.18.2 boot_1.3-27
## [70] bitops_1.0-7 Rdpack_2.1.2 rlang_0.4.10
## [73] pkgconfig_2.0.3 evaluate_0.14 purrr_0.3.4
## [76] labeling_0.4.2 htmlwidgets_1.5.3 tidyselect_1.1.1
## [79] magrittr_2.0.1 R6_2.5.1 generics_0.1.0
## [82] Hmisc_4.5-0 combinat_0.0-8 DBI_1.1.1
## [85] pillar_1.6.2 haven_2.4.1 foreign_0.8-81
## [88] withr_2.4.2 sp_1.4-5 abind_1.4-5
## [91] nnet_7.3-15 tibble_3.1.2 crayon_1.4.1
## [94] questionr_0.7.4 KernSmooth_2.23-20 utf8_1.2.1
## [97] tzdb_0.1.2 rmarkdown_2.10 jpeg_0.1-9
## [100] forcats_0.5.1 vcd_1.4-8 digest_0.6.27
## [103] xtable_1.8-4 httpuv_1.6.2 unmarked_1.1.1
## [106] stats4_4.0.5 munsell_0.5.0 bslib_0.2.5.1