Specificity, expression abundance and coefficient of variation of individual RGs
In the present study, an effort was made to identify the appropriate RGs in all the major livestock species that are native of Leh-ladakh region viz., Ladakhi cattle (LAC), Ladakhi yak (LAY), Ladakhi donkey (LAD), Double hump camel (DHC), Changthangi goat (CHG), Zanskar ponies (ZAP). The specificity of each primer pair was confirmed by the specific amplification checked in agarose gel and presence of single peak in melt curve analysis. The correlation coefficient (R2) and amplification efficiency (E) for individual primer pair in each of the six livestock species are given in Table 1. The expression abundance of individual RGs in each species is shown in Box Whisker plot (Fig. 2A-2F). The Ct values of individual RGs ranged from RPS23 (13.94) to HPRT1 (30.18) in LAC; RPS23 (14.37) to RPS15 (33.82) in LAY; RPS23 (13.86) to RPS15 (35.47) in LAD; RPS15 (16.05) to RPS23 (34.90) in DHC; RPS15 (13.63) to RPS23 (34.92) in CHG; RPS15 (16.06) to RPS23 (36.06) in ZAP (Table 2).
Table 1 Gene symbol, primer sequence, melting temperature (Ta), amplicon size, slope, PCR efficiency and R2 of RGs for each evaluated RG
Gene Symbol
|
Primers 5'-3' (Forward, Reverse)
|
Ta(ᵒC)
|
Amplicon Size (bp)
|
Slope
|
PCR efficiency
|
R2
|
Beta-Actin (ACTB)
|
F:5’GCGTGGCTACAGCTTCACC3’
R:3’TTGATGTCACGGACGATTTC5’
|
60
|
56
|
-3.10
|
107.40
|
0.997
|
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
|
F:5’TGGAAAGGCCATCACCATT3’
R:3’CCCACTTGATGTTGGCAG5’
|
60
|
60
|
-2.99
|
119.28
|
0.997
|
Eukaryotic translation elongation factor 1 alpha 1(EEF1A1)
|
F:5’CATCCCAGGCTGACTGTGC3’
R:3’TGTAAGCCAAAAGGGCATG5’
|
60
|
101
|
-3.11
|
109.65
|
0.998
|
β2 Microglobulin (B2M)
|
F:5’CTGCTATGTGTATGGGTTCC3’
R:3’GGAGTGAACTCAGCGTG5’
|
60
|
101
|
-3.03
|
114.64
|
0.999
|
Ribosomal protein L4 (RPL4)
|
F:5’TTGGAAACATGTGTCGTGG3’
R:3’GCAGATGGCGTATCGCTTCT5’
|
60
|
101
|
-3.12
|
109.45
|
0.998
|
Ribosomal protein S15 (RPS15)
|
F:5’GAATGGTGCGCATGAATGT3’
R:3’GACTTTGGAGCACGGCCTA5’
|
60
|
101
|
-2.89
|
127.12
|
0.996
|
Ribosomal protein S15 (RPS23)
|
F:5’CCCAATGATGGTTGCTTGAA3’
R:3’CGGACTCCAGGAATGTCAC5’
|
60
|
101
|
-3.20
|
102.27
|
0.990
|
Ribosomal protein S9 (RPS9)
|
F:5’CCTCGACCAAGAGCTGAAG3’
R:3’CCTCCAGACCTCACGTTTGT5’
|
60
|
54
|
-3.03
|
113.54
|
0.996
|
Ubiquitously
expressed transcript (UXT)
|
F:5’TGTGGCCCTTGGATATGGTT3’
R:3’GGTTGTCGCTGAGCTCTGTG5’
|
60
|
101
|
-3.33
|
99.36
|
0.988
|
Hypoxanthine Phosphoribosyl transferase (HPRT1)
|
F:5’GAGAAGTCCGAGTTGAGTT3’
R:3’GGCTCGTAGTGCAAATGAA5’
|
60
|
101
|
-3.03
|
113.60
|
0.988
|
Table 2 The average raw Ct values of individual RGs in different species
S.No
|
SAMPLE
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
1
|
LAC 1
|
22.166
|
24.350
|
22.395
|
18.810
|
19.511
|
15.934
|
26.065
|
21.560
|
19.355
|
30.175
|
2
|
LAC 2
|
21.865
|
22.880
|
20.150
|
17.320
|
19.785
|
14.275
|
23.860
|
20.055
|
18.195
|
28.375
|
3
|
LAC 3
|
22.080
|
23.515
|
19.975
|
16.895
|
19.550
|
14.240
|
24.175
|
19.665
|
18.715
|
27.950
|
4
|
LAC 4
|
22.295
|
24.660
|
19.865
|
17.260
|
19.570
|
14.094
|
24.545
|
20.145
|
18.565
|
28.135
|
5
|
LAC 5
|
22.525
|
22.640
|
20.035
|
17.090
|
19.205
|
13.943
|
24.675
|
19.605
|
18.745
|
28.865
|
6
|
LAC 6
|
22.065
|
24.900
|
20.410
|
17.225
|
19.445
|
14.062
|
24.505
|
19.750
|
17.735
|
29.690
|
|
|
|
|
|
|
|
|
|
|
|
|
7
|
LAY 11
|
18.984
|
22.124
|
22.675
|
23.616
|
33.537
|
17.509
|
22.220
|
23.867
|
22.913
|
24.790
|
8
|
LAY 12
|
17.341
|
20.518
|
21.299
|
20.124
|
29.057
|
15.891
|
22.872
|
24.407
|
20.860
|
24.951
|
9
|
LAY 15
|
17.458
|
19.617
|
21.063
|
19.573
|
30.821
|
14.365
|
22.848
|
25.646
|
18.109
|
24.333
|
10
|
LAY 16
|
18.306
|
21.148
|
22.322
|
23.314
|
30.667
|
15.700
|
23.041
|
23.709
|
22.436
|
25.037
|
11
|
LAY 18
|
19.202
|
21.497
|
23.420
|
23.584
|
33.815
|
16.677
|
22.838
|
24.405
|
22.954
|
25.410
|
12
|
LAY 19
|
18.548
|
20.981
|
22.407
|
23.161
|
33.664
|
16.822
|
22.763
|
23.866
|
21.809
|
25.026
|
|
|
|
|
|
|
|
|
|
|
|
|
13
|
LAD 1
|
22.631
|
21.085
|
19.703
|
18.913
|
34.072
|
14.334
|
23.918
|
21.676
|
17.632
|
25.613
|
14
|
LAD 2
|
24.527
|
20.870
|
20.745
|
20.654
|
31.857
|
14.842
|
22.433
|
21.309
|
18.578
|
26.040
|
15
|
LAD 3
|
20.735
|
20.623
|
19.752
|
17.940
|
33.395
|
14.630
|
23.017
|
23.167
|
16.685
|
24.795
|
16
|
LAD 4
|
23.579
|
19.967
|
22.779
|
19.457
|
34.096
|
14.027
|
22.706
|
21.676
|
18.105
|
25.826
|
17
|
LAD 5
|
22.868
|
20.767
|
24.162
|
20.442
|
35.473
|
13.857
|
21.455
|
20.553
|
17.750
|
25.568
|
|
|
|
|
|
|
|
|
|
|
|
|
18
|
CHG 27
|
14.350
|
18.640
|
19.792
|
18.836
|
34.923
|
13.633
|
21.406
|
23.870
|
28.316
|
25.075
|
19
|
CHG 28
|
15.877
|
18.359
|
19.575
|
20.644
|
30.897
|
15.230
|
20.443
|
20.810
|
33.046
|
25.448
|
20
|
CHG 29
|
17.404
|
17.562
|
19.358
|
18.338
|
34.623
|
15.027
|
22.621
|
23.095
|
32.082
|
24.355
|
21
|
CHG 30
|
16.321
|
18.014
|
19.434
|
20.044
|
33.763
|
14.099
|
20.786
|
20.015
|
31.783
|
24.553
|
22
|
CHG 33
|
15.238
|
17.090
|
19.151
|
17.746
|
30.183
|
13.943
|
21.771
|
22.071
|
26.700
|
24.858
|
|
|
|
|
|
|
|
|
|
|
|
|
23
|
DHC 34
|
23.103
|
18.367
|
19.258
|
17.584
|
30.718
|
17.048
|
18.195
|
18.228
|
20.174
|
21.929
|
24
|
DHC 35
|
22.565
|
19.002
|
19.688
|
17.167
|
33.965
|
16.055
|
20.889
|
22.323
|
21.401
|
22.778
|
25
|
DHC 37
|
20.412
|
19.155
|
18.550
|
17.387
|
31.888
|
16.886
|
19.762
|
20.840
|
19.244
|
22.105
|
26
|
DHC 38
|
24.128
|
19.254
|
19.583
|
17.930
|
34.898
|
17.266
|
19.434
|
18.681
|
20.421
|
22.271
|
27
|
DHC 39
|
22.619
|
19.997
|
20.835
|
16.869
|
30.624
|
17.983
|
20.106
|
21.816
|
20.867
|
22.271
|
|
|
|
|
|
|
|
|
|
|
|
|
28
|
ZAP 1
|
23.198
|
16.494
|
21.355
|
20.253
|
34.904
|
18.130
|
27.172
|
19.203
|
16.340
|
22.058
|
29
|
ZAP 2
|
19.785
|
16.688
|
21.265
|
19.610
|
36.059
|
17.718
|
27.222
|
18.761
|
16.059
|
25.723
|
30
|
ZAP 3
|
20.695
|
18.157
|
21.454
|
20.502
|
36.064
|
17.868
|
27.144
|
19.211
|
20.486
|
26.809
|
31
|
ZAP 4
|
25.664
|
19.940
|
19.839
|
18.813
|
33.605
|
16.471
|
26.166
|
17.547
|
19.799
|
24.798
|
32
|
ZAP 5
|
19.687
|
16.401
|
21.097
|
19.991
|
34.567
|
17.589
|
27.407
|
18.887
|
16.074
|
25.422
|
Expression stability analysis of RGs in each livestock species
Ladakhi Cattle (LAC)
The geNorm analysis ranked candidate reference genes as per their mean expression stability value (M value) which was below the threshold value of 1.5 for all the 10 RGs. The ranking order based on M value were EEF1A1=RPL4> RPS23> RPS9> UXT> B2M> GAPDH> RPS15> HPRT1> ACTB (Fig 3A). The M value ranged from 0.147 (EEF1A1) to 0.689 (ACTB). The lower M value indicates higher expression stability while higher M value indicates lower expression stability. On the basis of M value, EEF1A1=RPL4 RG pair was most stable expressed while ACTB was least stable. Another parameter that was evaluated by geNorm was the pairwise variation Vn/n + 1 in order to calculate the optimal number of RGs to be required for normalization. The pairwise variation (V) score of all the RGs were below 0.15 (Fig. 3B) which is an ideal pairwise recommended score [35]. Therefore, as per V value, combination of two RGs could be suggested to normalize the qPCR data in PBMCs of Ladakhi cattle.
In NormFinder analysis as well, the ranking stability of individual RGs were decided by the lower values indicating higher stability. In LAC, Normfinder analysis resulted in same panel of stable RGs (EEF1A1, RPL4, UXT, RPS23,) as identified in geNorm analysis. On the other hand, ACTB, HPRT1, RPS15 RGs were identified as least stable. The ranking order from most to least stable RGs was as follows: EEF1A1>RPL4> UXT> RPS23> RPS9> B2M> GAPDH> RPS15> HPRT1> ACT (Table 3).
Table 3 Overall Ranking of Best suitable RGs across different species
Species
|
Ranking
|
geNorm
|
Normfinder
|
Bestkeeper
|
RefFinder
|
|
|
M value
|
Stability value
|
STDEV
|
coff. of corr
|
|
Ladakhi cattle (LAC)
|
1
|
EEF1A1(0.147)
|
EEF1A1 (0.171)
|
RPS15 (0.123)
|
EEF1A1 (0.978)
|
EEF1A1 (1.41)
|
2
|
RPL4(0.147)
|
RPL4 (0.278)
|
GAPDH (0.163)
|
RPS9 (0.973)
|
RPL4 (2.11)
|
3
|
RPS23 (0.186)
|
UXT (0.297)
|
B2M (0.391)
|
RPS23 (0.973)
|
RPS23 (3.98)
|
4
|
RPS9 (0.267)
|
RPS23 (0.301)
|
EEF1A1 (0.468)
|
RPL4 (0.959)
|
UXT (4.36)
|
5
|
UXT (0.322)
|
RPS9 (0.498)
|
RPL4 (0.481)
|
UXT (0.941)
|
RPS15 (4.76)
|
6
|
B2M (0.413)
|
B2M (0.502)
|
UXT (0.489)
|
HPRT1 (0.763)
|
B2M (5.05)
|
7
|
GAPDH (0.512)
|
GAPDH (0.597)
|
RPS23 (0.503)
|
B2M (0.637)
|
GAPDH (5.12)
|
8
|
RPS15 (0.563)
|
RPS15 (0.602)
|
RPS9 (0.642)
|
ACTB (0.433)
|
RPS9 (5.32)
|
9
|
HPRT1 (0.605)
|
HPRT1 (0.616)
|
HPRT1 (0.712)
|
GAPDH (0.017)
|
HPRT1 (9)
|
10
|
ACTB (0.689)
|
ACTB (0.922)
|
ACTB (0.812)
|
RPS15 (0.001)
|
ACTB (10)
|
|
Ladakhi
Yak
(LAY)
|
1
|
GAPDH (0.223)
|
GAPDH (0.112)
|
UXT (0.180)
|
GAPDH (0.967)
|
GAPDH (1.41)
|
2
|
RPS9 (0.223)
|
RPS9 (0.112)
|
HPRT1 (0.240)
|
RPS9 (0.965)
|
RPS9 (2.21)
|
3
|
ACTB (0.386)
|
ACTB (0.202)
|
RPL4 (0.500)
|
EEF1A1 (0.960)
|
ACTB (3.41)
|
4
|
RPS23 (0.507)
|
RPS23 (0.524)
|
GAPDH (0.600)
|
ACTB (0.929)
|
HPRT1 (3.76)
|
5
|
HPRT1 (0.595)
|
HPRT1 (0.595)
|
ACTB (0.610)
|
B2M (0.927)
|
UXT (3.83)
|
6
|
UXT (0.708)
|
UXT (1.025)
|
RPS9 (0.680)
|
RPS23 (0.886)
|
RPS23 (4.86)
|
7
|
B2M (0.892)
|
EEF1A1 (1.191)
|
RPS23 (0.840)
|
RPS15 (0.813)
|
RPL4 (7.02)
|
8
|
EEF1A1 (0.992)
|
B2M (1.254)
|
B2M (1.350)
|
HPRT1 (0.727)
|
EEF1A1 (7.71)
|
9
|
RPL4 (1.121)
|
RPS15 (1.523)
|
EEF1A1 (1.590)
|
UXT (0.001)
|
B2M (7.74)
|
10
|
RPS15 (1.242)
|
RPL4 (1.538)
|
RPS15 (1.750)
|
RPL4 (0.001)
|
RPS15 (9.74)
|
|
Ladakhi
Donkey
(LAD)
|
1
|
HPRT1(0.250)
|
HPRT1 (0.123)
|
ACTB (0.295)
|
HPRT1 (0.942)
|
HPRT1 (1.19)
|
2
|
B2M (0.250)
|
B2M (0.324)
|
HPRT1 (0.311)
|
GAPDH (0.941)
|
B2M (2.00)
|
3
|
RPS23 (0.571)
|
ACTB (0.518)
|
RPS23 (0.318)
|
B2M (0.940)
|
ACTB (2.45)
|
4
|
ACTB (0.612)
|
RPS23 (0.605)
|
B2M (0.472)
|
EEF1A1 (0.927)
|
RPS23 (3.46)
|
5
|
EEF1A1 (0.751)
|
EEF1A1 (0.851)
|
RPL4 (0.598)
|
RPS9 (0.619)
|
EEF1A1 (5.44)
|
6
|
GAPDH (0.857)
|
UXT (1.181)
|
UXT (0.613)
|
RPS15 (0.022)
|
UXT (6.24)
|
7
|
UXT (0.984)
|
GAPDH (1.208)
|
EEF1A1 (0.852)
|
ACTB (0.001)
|
RPL4 (7.11)
|
8
|
RPL4 (1.090)
|
RPL4 (1.366)
|
RPS15 (0.92)
|
RPS23 (0.001)
|
GAPDH (7.17)
|
9
|
RPS15 (1.233)
|
RPS15 (1.377)
|
GAPDH (0.95)
|
UXT (0.00)
|
RPS15 (8.74)
|
10
|
RPSP (1.405)
|
RPS9 (1.912)
|
RPS9 (1.635)
|
RPL4 (0.001)
|
RPS9 (10.00)
|
|
Chanthangi
Goat
(CHG)
|
1
|
RPS9(0.378)
|
RPS9 (0.310)
|
RPS9 (0.1760)
|
B2M (0.847)
|
RPS9 (1)
|
2
|
HPRT1 (0.378)
|
RPS23 (0.477)
|
HPRT1 (0.324)
|
RPS23 (0.676)
|
HPRT1 (2.38)
|
3
|
ACTB (0.434)
|
ACTB (0.486)
|
ACTB (0.486)
|
GAPDH (0.623)
|
ACTB (2.71)
|
4
|
RPS23 (0.636)
|
HPRT1 (0.740)
|
RPS23 (0.595)
|
RPS15 (0.580)
|
RPS23 (3.13)
|
5
|
EEF1A1 (0.803)
|
GAPDH (0.953)
|
UXT (0.633)
|
EEF1A1 (0.435)
|
UXT (5.48)
|
6
|
UXT (0.973)
|
UXT (1.000)
|
GAPDH (0.834)
|
ACTB (0.429)
|
GAPDH (5.96)
|
7
|
GAPDH (1.066)
|
EEF1A1 (1.120)
|
EEF1A1 (0.974)
|
RPS9 (0.422)
|
EEF1A1 (6.44)
|
8
|
RPL4 (1.254)
|
RPL4 (1.764))
|
RPL4 (1.248)
|
UXT (0.087)
|
RPL4 (8)
|
9
|
RPS15 (1.474)
|
RPS15 (2.015
|
RPS15 (1.869)
|
RPL4 (0.051)
|
RPS15 (9)
|
10
|
B2M (1.721)
|
B2M (2.517)
|
B2M (2.301)
|
HPRT1 (0.001)
|
B2M (10)
|
|
|
|
|
|
|
|
Double hump Camel
(DHC)
|
1
|
B2M (0.600)
|
HPRT1 (0.295)
|
HPRT1 (0.203)
|
GAPDH (0.372)
|
HPRT1 (1.32)
|
2
|
RPS9 (0.600)
|
ACTB (0.418)
|
EEF1A1 (0.294)
|
ACTB (0.677)
|
ACTB (2.63)
|
3
|
HPRT1 (0.664)
|
B2M (0.420)
|
ACTB (0.377)
|
RPS9 (0.751)
|
B2M (2.71)
|
4
|
ACTB (0.680)
|
RPS9 (0.586)
|
RPS23 (0.462)
|
EEF1A1 (0.001)
|
RPS9 (2.99)
|
5
|
RPS23 (0.747)
|
UXT (0.668)
|
RPS9 (0.542)
|
RPS15 (0.446)
|
EEF1A1(4.56)
|
6
|
EEF1A1 (0.794)
|
EEF1A1 (0.731)
|
B2M (0.572)
|
RPS23 (0.055)
|
RPS23 (5.60)
|
7
|
UXT (0.860)
|
RPS23 (0.915)
|
UXT (0.693)
|
UXT (0.774)
|
UXT (5.92)
|
8
|
GAPDH (0.992)
|
RPS15 (1.333)
|
GAPDH (0.862)
|
RPL4 (0.599)
|
GAPDH (8.00)
|
9
|
RPL4 (1.180)
|
RPL4 (1.766)
|
RPL4 (1.538)
|
B2M (0.809)
|
RPL4 (9.00)
|
10
|
RPS15 (1.352)
|
RPS15 (1.856)
|
RPS15 (1.61)
|
HPRT1 (0.797)
|
RPS15 (10.00)
|
|
|
|
|
|
|
|
Zanskar
Horse
(ZAP)
|
1
|
RPS9(0.135)
|
EEF1A1 (0.631)
|
UXT (0.341)
|
B2M (0.947)
|
RPS9 (1.73)
|
2
|
RPL4 (0.135)
|
UXT (0.684)
|
RPS23 (0.434)
|
ACTB (0.662)
|
RPL4 (2.51)
|
3
|
RPS23 (0.142)
|
RPS9 (0.749)
|
RPS9 (0.465)
|
HPRT1 (0.447)
|
UXT (2.66)
|
4
|
EEF1A1 (0.194)
|
RPS23 (0.759)
|
RPL4 (0.469
|
GAPDH (0.257)
|
EEF1A1 (2.78)
|
5
|
UXT (0.249)
|
RPL4 (0.763)
|
EEF1A1 (0.498)
|
RPS15 (0.182)
|
RPS23 (3.13)
|
6
|
RPS15 (0.411)
|
RPS15 (1.022)
|
RPS15 (0.816)
|
EEF1A1 (0.159)
|
RPS15 (6)
|
7
|
HPRT1 (0.824)
|
ACTB (1.36)
|
ACTB (1.211)
|
RPS9 (0.001)
|
ACTB (7.24)
|
8
|
ACTB (1.136)
|
HPRT1 (1.727)
|
HPRT1 (1.226)
|
RPS23 (0.001)
|
HPRT1 (7.74)
|
9
|
B2M (1.394)
|
B2M (1.945)
|
B2M (1.914)
|
UXT (0.001)
|
B2M (9)
|
10
|
GAPDH (1.712)
|
GAPDH (2.795)
|
GAPDH (20.99)
|
RPL4 (0.001)
|
GAPDH (10)
|
The gene expression variation for 10 candidate RGs was also calculated using Best-Keeper algorithm. In BestKeeper analysis, raw Ct values were used to evaluate stability of individual RGs based on their SD and CV values. The lower value indicates higher expression stability; however, the SD > 1 value indicates the reference gene is unstable and cannot be used for normalization. The RPS15 and GAPDH genes having lowest SD values of 0.123, 0.163 indicated expression stability. This was followed by B2M, EEF1A1, RPL4, UXT, RPS23, RPS9, HPRT1 and ACTB with SD values 0.391, 0.468, 0.481, 0.491, 0.503, 0.642, 0.712 and 0.812, respectively (Table 4). The ACTB gene on the other hand was least stable gene with highest SD value. Additionally, the inter-gene relationship for 10 RGs pairs was also estimated. Strong correlation coefficients (r) were observed for RPL4/EEF1A1 (0.980), EEF1A1/RPS9 (0.971), RPS23/RPS9 (0.966), RPS23/EEF1A1 (0.962), RPL4/RPS23 (0.961), RPL4/ RPS9 (0.922), UXT/ RPS9 (0.898) (Table 5). This analysis provided strong evidence that these pair of genes have similar expression pattern across the animals. Further Best-Keeper index was calculated for each gene and the correlation between each candidate RGs and Best-Keeper was estimated. The relationship between RGs and Best-Keeper was described in terms of Pearson correlation coefficient (r), coefficient of determination (r2) and the p value. The p<0.05 was obtained for all genes indicating a significant contribution of all genes towards the index. Though the EEF1A1 (0.978) and RPS9 (0.973) showed high correlation values but their high fold change makes these genes as unreliable reference genes. The statistically significant SD and correlation shown by the RGs from with BestKeeper algorithm appeared to be consistent with their evaluation assessed by geNorm and Normfinder.
Table 4 Analysis of parameters based quantitative cycling points (CP) for 10 candidate RGs in LAC
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
n
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
geo Mean [CP]
|
22.170
|
23.810
|
20.460
|
17.420
|
19.510
|
14.410
|
24.630
|
20.120
|
18.550
|
28.860
|
AR Mean [CP]
|
22.170
|
23.830
|
20.480
|
17.440
|
19.510
|
14.420
|
24.640
|
20.130
|
18.560
|
28.870
|
min [CP]
|
21.870
|
22.640
|
19.870
|
16.900
|
19.210
|
13.940
|
23.860
|
19.610
|
17.740
|
27.950
|
max [CP]
|
22.530
|
24.900
|
22.400
|
18.810
|
19.790
|
15.930
|
26.070
|
21.560
|
19.360
|
30.180
|
std dev [+/- CP]
|
0.16
|
0.81
|
0.64
|
0.46
|
0.12
|
0.50
|
0.49
|
0.48
|
0.39
|
0.71
|
CV [% CP]
|
0.740
|
3.410
|
3.130
|
2.630
|
0.630
|
3.490
|
1.980
|
2.390
|
2.110
|
2.470
|
min [x-fold]
|
-1.230
|
-2.250
|
-1.500
|
-1.440
|
-1.230
|
-1.380
|
-1.710
|
-1.430
|
-1.750
|
-1.870
|
max [x-fold]
|
1.280
|
2.130
|
3.840
|
2.610
|
1.210
|
2.880
|
2.710
|
2.710
|
1.750
|
2.500
|
std dev [+/- x-fold]
|
1.120
|
1.760
|
1.560
|
1.370
|
1.090
|
1.420
|
1.400
|
1.400
|
1.310
|
1.640
|
N = number of samples, geo Mean[CP] = geometric mean of CP; ar Mean[CP] = arithmetic mean of CP; min [CP] and max [CP] = extreme values of CP; Std dev [±CP] = standard deviation of the CP; CV [%CP] = coefficient of variation expressed as a percentage on the CP values; min [x-fold] and max [x-fold] = extreme values of expression levels expressed as absolute x-fold over or under coefficient; std dev[±x-fold] = standard deviation of the absolute regulation coefficients.
Table 5 Analysis of repeated pair-wise correlation amongst genes in LAC with BestKeeper index
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
|
ACTB
|
-0.107
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
p-value
|
0.84
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
RPS9
|
-0.08
|
0.309
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
p-value
|
0.88
|
0.552
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
EEF1A1
|
-0.056
|
0.316
|
0.971
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
p-value
|
0.916
|
0.542
|
0.001
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
RPS15
|
-0.856
|
0.072
|
-0.014
|
0.08
|
-
|
-
|
-
|
-
|
-
|
-
|
|
p-value
|
0.03
|
0.892
|
0.979
|
0.881
|
-
|
-
|
-
|
-
|
-
|
-
|
|
RPS23
|
-0.14
|
0.252
|
0.966
|
0.962
|
0.137
|
-
|
-
|
-
|
-
|
-
|
|
p-value
|
0.791
|
0.631
|
0.002
|
0.002
|
0.796
|
-
|
-
|
-
|
-
|
-
|
|
UXT
|
0.339
|
0.378
|
0.898
|
0.888
|
-0.344
|
0.849
|
-
|
-
|
-
|
-
|
|
p-value
|
0.511
|
0.46
|
0.015
|
0.018
|
0.504
|
0.033
|
-
|
-
|
-
|
-
|
|
RPL4
|
-0.102
|
0.34
|
0.922
|
0.98
|
0.206
|
0.961
|
0.84
|
-
|
-
|
-
|
|
p-value
|
0.848
|
0.51
|
0.009
|
0.001
|
0.695
|
0.002
|
0.036
|
-
|
-
|
-
|
|
B2M
|
0.396
|
-0.148
|
0.585
|
0.611
|
-0.197
|
0.698
|
0.702
|
0.652
|
-
|
-
|
|
p-value
|
0.436
|
0.779
|
0.222
|
0.197
|
0.709
|
0.123
|
0.12
|
0.16
|
-
|
-
|
|
HPRT1
|
0.052
|
0.4
|
0.82
|
0.747
|
-0.317
|
0.645
|
0.775
|
0.612
|
0.162
|
-
|
|
p-value
|
0.922
|
0.432
|
0.046
|
0.088
|
0.541
|
0.166
|
0.07
|
0.197
|
0.759
|
-
|
|
|
BestKeeper vs.
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
|
coeff. of corr. [r]
|
0.017
|
0.433
|
0.973
|
0.978
|
0.001
|
0.959
|
0.941
|
0.959
|
0.637
|
0.763
|
|
p-value
|
0.975
|
0.392
|
0.001
|
0.001
|
0.971
|
0.003
|
0.005
|
0.003
|
0.173
|
0.078
|
|
Additionally, RefFinder based analysis was carried out that ranks the stability order of RGs in a more refined way by taking into consideration geNorm, NormFinder, BestKeeper, and delta Ct algorithms. The stability order and ranking of the RGs as per RefFinder were; EEF1A1 (1.41) RPL4 (2.11), RPS23 (3.98), UXT (4.36), RPS15 (4.76), B2M (5.05), GAPDH (5.12), RPS9 (5.32), HPRT1 (9), ACTB (10).
Ladakhi Yak (LAY)
The M value for all the 10 genes in geNorm analysis were found to be within acceptable range in LAY. The ranking order of RGs was GAPDH=RPS9> ACTB> RPS23> HPRT1> UXT> B2M> EEF1A1> RPL4> RPS15 (Fig. 3D). GAPDH and RPS9 showed higher gene expression stability with M value of 0.223 followed by ACTB, RPS23 and HPRT1 with M value of 0.386, 0.507, 0.595 respectively (Table 3). On the other hand, RPS15, RPL4 and EEF1A1 were least stable with higher M values of 1.242, 1.121 and 0.992, respectively. The pair wise variation analysis showed V4/5 combination with least V value (0.132) followed by V3/4 (0.148) and V5/V6 (0.150) combinations (Fig. 3E). Since all these V values were well within the acceptable range (recommended cut-off value 0.15), therefore use of panel of 3 RGs (GAPDH, RPS9 and ACTB) is likely to provide most accurate normalization in Ladakhi yak samples. The Normfinder analysis also identified same set of RGs in LAY samples with highest stability; GAPDH (0.112), RPS9 (0.112) and ACTB (0.202) albeit slight change in their ranking order; GAPDH> RPS9> ACTB>RPS23> HPRT1> UXT> EEF1A1> B2M> RPS15> RPL4 (Table 3). Similar to geNorm, RPL4 (1.538) and RPS15 (1.523) were found to be least stable RGs.
In BestKeeper analysis, UXT was found to be most stable with minimum SD value (0.180) followed by HPRT1, RPL4, GAPDH, ACTB, RPS9, RPS23, B2M, EEF1A1, RPS15 with the SD values of 0.240, 0.500, 0.600, 0.610, 0.680, 0.840, 1.350, 1.590, 1.750, respectively (Table 6). Additionally, high correlation coefficient was observed for RPS9/GAPDH (r=0.973), B2M/ACTB (r=0.942), EEF1A1/GAPDH (r=0.931), EEF1A1/RPS9 (r=0.923), RPS23/ACTB (r=0.914) and B2M/EEF1A1 (r=0.909) pair combinations. The best correlation between RGs and BestKeeper was observed for GAPDH (r=0.967), RPS9 (r=0.965), EEF1A1 (r=0.960), ACTB (r=0.929), B2M (r=0.927) (Table 7). The high correlation values for these genes indicated their reliability as RGs, The GAPDH, RPS9 and ACTB were termed as best RGs on the basis of highest correlation value and less SD.
Table 6 Analysis of parameters based quantitative cycling points (CP) for 10 candidate RGs in LAY
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
n
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
6
|
geo Mean [CP]
|
18.29
|
20.97
|
22.18
|
22.16
|
31.87
|
16.13
|
22.76
|
24.31
|
21.44
|
24.92
|
AR Mean [CP]
|
18.31
|
20.98
|
22.2
|
22.23
|
31.93
|
16.16
|
22.76
|
24.32
|
21.51
|
24.93
|
min [CP]
|
17.34
|
19.62
|
21.06
|
19.57
|
29.06
|
14.37
|
22.22
|
23.71
|
18.11
|
24.33
|
max [CP]
|
19.2
|
22.12
|
23.42
|
23.62
|
33.82
|
17.51
|
23.04
|
25.65
|
22.95
|
25.41
|
std dev [+/- CP]
|
0.60
|
0.61
|
0.68
|
1.59
|
1.75
|
0.84
|
0.18
|
0.50
|
1.35
|
0.24
|
CV [% CP]
|
3.3
|
2.9
|
3.06
|
7.14
|
5.47
|
5.21
|
0.8
|
2.06
|
6.29
|
0.98
|
min [x-fold]
|
-1.94
|
-2.54
|
-2.18
|
-6.02
|
-7.04
|
-3.39
|
-1.46
|
-1.52
|
-10.08
|
-1.51
|
max [x-fold]
|
1.88
|
2.22
|
2.36
|
2.75
|
3.85
|
2.6
|
1.21
|
2.53
|
2.84
|
1.4
|
std dev [+/- x-fold]
|
1.52
|
1.52
|
1.6
|
3.01
|
3.35
|
1.79
|
1.13
|
1.42
|
2.55
|
1.18
|
Table 7 Analysis of repeated pair-wise correlation amongst genes in LAY with BestKeeper index
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
ACTB
|
0.859
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.028
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS9
|
0.973
|
0.84
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.001
|
0.037
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
EEF1A1
|
0.931
|
0.887
|
0.923
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.007
|
0.019
|
0.009
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS15
|
0.892
|
0.632
|
0.805
|
0.751
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.017
|
0.178
|
0.053
|
0.085
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS23
|
0.784
|
0.914
|
0.755
|
0.786
|
0.693
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.065
|
0.011
|
0.083
|
0.064
|
0.127
|
-
|
-
|
-
|
-
|
-
|
UXT
|
-0.437
|
-0.581
|
-0.265
|
-0.31
|
-0.525
|
-0.652
|
-
|
-
|
-
|
-
|
p-value
|
0.386
|
0.227
|
0.611
|
0.55
|
0.285
|
0.161
|
-
|
-
|
-
|
-
|
RPL4
|
-0.565
|
-0.792
|
-0.597
|
-0.799
|
-0.322
|
-0.761
|
0.207
|
-
|
-
|
-
|
p-value
|
0.243
|
0.06
|
0.211
|
0.056
|
0.533
|
0.079
|
0.694
|
-
|
-
|
-
|
B2M
|
0.82
|
0.942
|
0.87
|
0.909
|
0.538
|
0.858
|
-0.292
|
-0.879
|
-
|
-
|
p-value
|
0.046
|
0.005
|
0.024
|
0.012
|
0.271
|
0.029
|
0.574
|
0.021
|
-
|
-
|
HPRT1
|
0.62
|
0.602
|
0.777
|
0.671
|
0.371
|
0.587
|
0.186
|
-0.634
|
0.81
|
-
|
p-value
|
0.19
|
0.206
|
0.069
|
0.145
|
0.47
|
0.221
|
0.724
|
0.176
|
0.051
|
-
|
BestKeeper vs.
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
coeff. of corr. [r]
|
0.967
|
0.929
|
0.965
|
0.96
|
0.813
|
0.886
|
0.001
|
0.001
|
0.927
|
0.727
|
p-value
|
0.002
|
0.007
|
0.002
|
0.002
|
0.049
|
0.019
|
0.401
|
0.096
|
0.008
|
0.102
|
The RefFinder analysis also identified GAPDH, RPS9 and ACTB to be most stable RGs while RPS15, B2M and EEF1A1 were the least stable RGS in LAY. In the present investigation, all four methods geNorm, Normfinder BestKeeper and RefFinder have demonstrated that GAPDH, RPS9 and ACTB are the most stable RGs in PBMCs of LAY.
Ladakhi Donkey (LAD)
In Ladakhi donkey as well, the geNorm analysis showed mean expression stability values of 10 RGs within the acceptable range and varied from 0.250 (HPRT1= B2M) to 1.405 (RPS9) (Table 3). The stability ranking of RGs was: HPRT1=B2M> RPS23> ACTB> EEF1A1> GAPDH> UXT> RPL4> RPS15> RPS9 (Fig. 3G). The B2M and HPRT1 RGs showed highest expression stability with lowest M value while RPS9 and RPS15 RGs showed least expression stability with highest M value. Based on pair-wise variation analysis (V value), V3/4 combination (B2M HPRT1 and RPS23) with V value of 0.142 was found to provide the most accurate normalization in Ladakhi donkey (Fig. 3H). In Normfinder analysis as well; HPRT1 (0.123), B2M (0.324) and ACTB (0.518) were most stable with lowest values (Fig. 3I). On the other hand, the RPS9 (1.912), RPS15 (1.377), and RPL4 (1.366) RGs on the other hand were least stable.
The BestKeeper analysis showed ACTB gene to be most stable with the lowest crossing point SD value of 0.295. This was followed by HPRT1, RPS23 and B2M RGs with SD value of 0.311, 0.318, and 0.472, respectively. On the other hand, RPS9 with highest crossing point SD value of 1.635 was found to be the least stable (Table 8). In addition, the inter-gene relation for 10 RGs pairs was also estimated. B2M/GAPDH (r=1.0), HPRT1/B2M (r=0.985), HPRT1/GAPDH (r=0.985), B2M/EEF1A1 (r=0.855) and EEF1A1/GAPDH (r=0.854) showed the strong correlation coefficients (Table 9). The highly correlated RGs were combined into BestKeeper index, and the correlation between each candidate RGs and BestKeeper was estimated. The relationship between RG and BestKeeper was described in terms of Pearson correlation coefficient (r), coefficient of determination correlation between BestKeeper and RGs was observed for HPRT1 (r=0.942) and GAPDH (r=0.941) followed by B2M (0.940) and EEF1A1 (0.927) genes. The statistically significant correlation shown by RGs (HPRT1, B2M) with the BestKeeper index appeared to be consistent with their evaluation as assessed by geNorm and Normfinder. RefFinder was another tool, were evaluating and identified RGs from comprehensive data set. HPRT1, B2M and ACTB were most stable and RPS9, RPS15 and GAPDH were least stable genes identified by RefFinder in LAD.
Table 8 Analysis of parameters based quantitative cycling points (CP) for 10 candidate RGs in LAD
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
n
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
geo Mean [CP]
|
22.83
|
20.66
|
21.35
|
19.45
|
33.76
|
14.33
|
22.69
|
21.66
|
17.74
|
25.56
|
AR Mean [CP]
|
22.87
|
20.66
|
21.43
|
19.48
|
33.78
|
14.34
|
22.71
|
21.68
|
17.75
|
25.57
|
min [CP]
|
20.73
|
19.97
|
19.7
|
17.94
|
31.86
|
13.86
|
21.45
|
20.55
|
16.69
|
24.79
|
max [CP]
|
24.53
|
21.09
|
24.16
|
20.65
|
35.47
|
14.84
|
23.92
|
23.17
|
18.58
|
26.04
|
std dev [+/- CP]
|
0.95
|
0.30
|
1.64
|
0.85
|
0.92
|
0.32
|
0.61
|
0.60
|
0.47
|
0.31
|
CV [% CP]
|
4.16
|
1.43
|
7.63
|
4.37
|
2.72
|
2.22
|
2.7
|
2.76
|
2.66
|
1.22
|
min [x-fold]
|
-4.3
|
-1.61
|
-3.15
|
-2.86
|
-3.73
|
-1.39
|
-2.36
|
-2.16
|
-2.07
|
-1.71
|
max [x-fold]
|
3.24
|
1.35
|
6.99
|
2.29
|
3.27
|
1.42
|
2.34
|
2.85
|
1.79
|
1.39
|
std dev [+/- x-fold]
|
1.93
|
1.23
|
3.11
|
1.81
|
1.89
|
1.25
|
1.53
|
1.51
|
1.39
|
1.24
|
Table 9 Analysis of repeated pair-wise correlation amongst genes in LAD with BestKeeper index
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
ACTB
|
-0.067
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.915
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS9
|
0.345
|
-0.434
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.57
|
0.465
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
EEF1A1
|
0.854
|
0.096
|
0.608
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.065
|
0.878
|
0.276
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS15
|
-0.301
|
-0.134
|
0.629
|
-0.036
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.623
|
0.83
|
0.256
|
0.954
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS23
|
-0.004
|
0.363
|
-0.793
|
-0.171
|
-0.931
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.995
|
0.548
|
0.109
|
0.783
|
0.022
|
-
|
-
|
-
|
-
|
-
|
UXT
|
-0.281
|
0.204
|
-0.827
|
-0.674
|
-0.289
|
0.372
|
-
|
-
|
-
|
-
|
p-value
|
0.647
|
0.742
|
0.084
|
0.212
|
0.638
|
0.538
|
-
|
-
|
-
|
-
|
RPL4
|
-0.71
|
-0.162
|
-0.709
|
-0.895
|
-0.353
|
0.51
|
0.581
|
-
|
-
|
-
|
p-value
|
0.179
|
0.795
|
0.18
|
0.04
|
0.56
|
0.381
|
0.304
|
-
|
-
|
-
|
B2M
|
1
|
-0.065
|
0.343
|
0.855
|
-0.304
|
0
|
-0.282
|
-0.709
|
-
|
-
|
p-value
|
0.001
|
0.918
|
0.572
|
0.065
|
0.619
|
1
|
0.646
|
0.18
|
-
|
-
|
HPRT1
|
0.985
|
-0.04
|
0.339
|
0.814
|
-0.206
|
-0.094
|
-0.191
|
-0.739
|
0.985
|
-
|
p-value
|
0.002
|
0.949
|
0.577
|
0.094
|
0.74
|
0.881
|
0.758
|
0.153
|
0.002
|
-
|
|
BestKeeper vs.
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
coeff. of corr. [r]
|
0.941
|
0.001
|
0.619
|
0.927
|
0.022
|
0.001
|
0.001
|
0.001
|
0.94
|
0.942
|
p-value
|
0.017
|
0.863
|
0.265
|
0.024
|
0.972
|
0.612
|
0.406
|
0.046
|
0.018
|
0.017
|
Chanthangi Goat (CHG)
The geNorm analysis of all the 10 candidate RGs in Changthangi goat exhibited mean expression stability (M) values well below 1.5 (Table 3). The stability ranking RGs were in the following order; RPS9=HPRT>ACTB>RPS23>EEF1A1>UXT >GAPDH>RPL4>RPS15>B2M (Fig. 3J). The RPS9 and HPRT were most stable with lowest M value of 0.378 while RPS 15 and B2M had maximum expression variability and highest M values of 1.474 and 1.721, respectively.
Further, the pair-wise variation analysis provided within the acceptable limit on sequential addition of another gene to the two most stably expressed genes, viz., B2M and HPRT1, the pair-wise combination V2/3 gave the acceptable V value of 0.143 (<0.15) suggesting that the geometric mean between RPS9, HPRT1 and ACTB is optimal for data normalization in Changthangi goat (Fig. 3K). Similar to geNorm, Normfinder also identified RPS9 (0.310), RPS23 (0.477), ACTB (0.486) and HPRT1 (0.740) as most stable and B2M (2.517) and RPS15 (2.015) as least stably expressed genes (Table 3). There was good agreement between geNorm and Normfinder outcome, albeit slight variation was observed in the ranking of RGs. The BestKeeper algorithm showed consistent expression levels for all the RGs.
RPS9 (0.176), exhibited low SD and 0.422 correlation coefficients in BestKeeper analysis, pointing towards their expression stability (Table 10). Additionally, intergene relationship were identified in RGs. RPS9/ACTB (r=0.974), B2M/RPS23 (r=0.801), B2M/EEF1A1 (r=0.739), and RPS23/GAPDH (r=0.712) showed the strong correlation coefficients (Table 11). B2M (0.847) showed the high correlation value but they showed the high fold change thus their reliability as a RGs is not applicable. RefFinder were identified the overall ranking of the gene. The ranking of genes was RPS9 (1), HPRT1 (2.38), ACTB (2.71), RPS23 (3.13), UXT (5.48), GAPDH (5.96), EEF1A1 (6.44), RPL4 (8), RPS15 (9), B2M (10). In the present investigation, all four algorithmic methods geNorm Normfinder, BestKeeper and RefFinder have demonstrated that RPS9, HPRT1 and ACTB are the most stable RGs in CHG.
Table 10 Analysis of parameters based quantitative cycling points (CP) for 10 candidate RGs in CHG
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
n
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
geo Mean [CP]
|
15.8
|
17.92
|
19.46
|
19.09
|
32.82
|
14.37
|
21.39
|
21.92
|
30.29
|
24.86
|
AR Mean [CP]
|
15.84
|
17.93
|
19.46
|
19.12
|
32.88
|
14.39
|
21.41
|
21.97
|
30.39
|
24.86
|
min [CP]
|
14.35
|
17.09
|
19.15
|
17.75
|
30.18
|
13.63
|
20.44
|
20.01
|
26.7
|
24.36
|
max [CP]
|
17.4
|
18.64
|
19.79
|
20.64
|
34.92
|
15.23
|
22.62
|
23.87
|
33.05
|
25.45
|
std dev [+/- CP]
|
0.83
|
0.49
|
0.18
|
0.97
|
1.87
|
0.6
|
0.63
|
1.25
|
2.3
|
0.32
|
CV [% CP]
|
5.27
|
2.71
|
0.9
|
5.1
|
5.68
|
4.14
|
2.96
|
5.68
|
7.57
|
1.3
|
min [x-fold]
|
-2.74
|
-1.78
|
-1.24
|
-2.54
|
-6.22
|
-1.67
|
-1.94
|
-3.77
|
-12
|
-1.41
|
max [x-fold]
|
3.02
|
1.64
|
1.26
|
2.92
|
4.3
|
1.81
|
2.34
|
3.85
|
6.8
|
1.51
|
std dev [+/- x-fold]
|
1.78
|
1.4
|
1.13
|
1.96
|
3.65
|
1.51
|
1.55
|
2.38
|
4.93
|
1.25
|
Table 11 Analysis of repeated pair-wise correlation amongst genes in CHG with BestKeeper index
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
ACTB
|
-0.379
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.53
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS9
|
-0.431
|
0.974
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.468
|
0.005
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
EEF1A1
|
0.096
|
0.663
|
0.482
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.878
|
0.222
|
0.412
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS15
|
0.163
|
0.422
|
0.522
|
-0.041
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.793
|
0.479
|
0.367
|
0.948
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS23
|
0.712
|
-0.035
|
-0.11
|
0.398
|
-0.185
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.177
|
0.956
|
0.86
|
0.507
|
0.766
|
-
|
-
|
-
|
-
|
-
|
UXT
|
0.341
|
-0.574
|
-0.393
|
-0.852
|
0.335
|
-0.009
|
-
|
-
|
-
|
-
|
p-value
|
0.574
|
0.311
|
0.513
|
0.067
|
0.581
|
0.988
|
-
|
-
|
-
|
-
|
RPL4
|
-0.293
|
0.05
|
0.274
|
-0.673
|
0.441
|
-0.264
|
0.689
|
-
|
-
|
-
|
p-value
|
0.632
|
0.937
|
0.655
|
0.213
|
0.457
|
0.668
|
0.199
|
-
|
-
|
-
|
B2M
|
0.697
|
0.334
|
0.208
|
0.739
|
0.194
|
0.801
|
-0.292
|
-0.475
|
-
|
-
|
p-value
|
0.191
|
0.583
|
0.737
|
0.154
|
0.754
|
0.103
|
0.633
|
0.419
|
-
|
-
|
HPRT1
|
-0.625
|
0.533
|
0.478
|
0.456
|
-0.494
|
0.081
|
-0.669
|
-0.08
|
-0.034
|
-
|
p-value
|
0.26
|
0.355
|
0.415
|
0.44
|
0.398
|
0.897
|
0.217
|
0.898
|
0.956
|
-
|
|
BestKeeper vs.
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
coeff. of corr. [r]
|
0.623
|
0.429
|
0.422
|
0.435
|
0.58
|
0.676
|
0.087
|
0.051
|
0.847
|
0.001
|
p-value
|
0.262
|
0.471
|
0.479
|
0.464
|
0.305
|
0.211
|
0.89
|
0.935
|
0.07
|
0.783
|
Double hump Camel (DHC)
The geNorm analysis of 10 RGs showed M values ranging from 0.600 to 1.352 in double hump camel (Table 3). The M values for all the RGs were within the acceptable limit of <1.5. On the basis of relative expression stability and stepwise exclusion, the ranking order of RGs was: B2M=RPS9 > HPRT1> ACTB> RPS23> EEF1A1> UXT> GAPDH> RPL4> RPS15 (Fig. 3M). The expression of RPS9 and B2M RGs with lowest M values of 0.600 were found to be most stable while RPL4 and RPS15 RGs with highest M values of 1.180 and 1.352, respectively were found to be least stable RGs in DHC. Based on pair-wise combination, the V values for V3/4, V5/6 and V6/7 and were close to the threshold value of 0.15. Therefore, the combination of V3/4 with ACTB, RPS9 and B2M RGs should provide the accurate normalization of qPCR data in DHC.
In Normfinder analysis, the RGs were ranked as follows: HPRT1> ACTB> B2M> RPS9> UXT> EEF1A1> RPS23> GAPDH> RPL4> RPS15 (Fig. 3O). The HPRT1 (0.295), ACTB (0.418), B2M (0.420), RPS9 (0.586) were four most stable RGs as per stability values.
In BestKeeper analysis, HPRT1 gene with the lowest crossing point SD value of 0.203 was found to be most stable. This was followed by EEF1A1, ACTB and RPS23 genes with SD values of 0.294, 0.377, and 0.462, respectively (Table12). On the other hand, RPS15, RPL4 and GAPDH RGs with high crossing point SD values of 1.61, 1.54, 0.86 respectively were found to be least stable. Strong correlation was observed in inter gene relationship of the RGs RPL4/UXT (r=0.908), HPRT1/UXT (r=0.884) and HPRT1/B2M (r=0.755) (Table 13). The relationship between RGs and BestKeeper was described in terms of Pearson correlation coefficient (r), coefficient of determination correlation between BestKeeper and RGs was observed for HPRT1 (r=0.797) and B2M (r=0.809) followed by UXT, RPS9 and ACTB gene.
ReFinder based overall analysis resulted in stability ranking of RGs as; HPRT1 (1.32) > ACTB (2.63) >, B2M (2.71) >, RPS9 (2.99) > EEF1A1, RPS23 (5.60) >UXT (5.92)> GAPDH (8.00)> RPL4 (9.00)> RPS15 (10.00). Overall, HPRT1, B2M and ACTB were identified as the most appropriate RGs in high altitude adapted DHC using all four algorithms.
Table 12 Analysis of parameters based quantitative cycling points (CP) for 10 candidate RGs in DHC
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
n
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
geo Mean [CP]
|
22.53
|
19.15
|
19.57
|
17.38
|
32.37
|
17.04
|
19.66
|
20.31
|
20.41
|
22.27
|
AR Mean [CP]
|
22.57
|
19.16
|
19.58
|
17.39
|
32.42
|
17.05
|
19.68
|
20.38
|
20.42
|
22.27
|
min [CP]
|
20.41
|
18.37
|
18.55
|
16.87
|
30.62
|
16.05
|
18.19
|
18.23
|
19.24
|
21.93
|
max [CP]
|
24.13
|
20
|
20.83
|
17.93
|
34.9
|
17.98
|
20.89
|
22.32
|
21.4
|
22.78
|
std dev [+/- CP]
|
0.86
|
0.38
|
0.54
|
0.29
|
1.61
|
0.46
|
0.69
|
1.54
|
0.57
|
0.2
|
CV [% CP]
|
3.82
|
1.97
|
2.77
|
1.69
|
4.97
|
2.71
|
3.52
|
7.55
|
2.8
|
0.91
|
min [x-fold]
|
-4.35
|
-1.72
|
-2.03
|
-1.43
|
-3.37
|
-1.98
|
-2.76
|
-4.23
|
-2.25
|
-1.27
|
max [x-fold]
|
3.03
|
1.8
|
2.4
|
1.46
|
5.77
|
1.92
|
2.35
|
4.03
|
1.99
|
1.42
|
std dev [+/- x-fold]
|
1.82
|
1.3
|
1.46
|
1.23
|
3.05
|
1.38
|
1.62
|
2.9
|
1.49
|
1.15
|
Table 13 Analysis of repeated pair-wise correlation amongst genes in DHC with BestKeeper index
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
ACTB
|
-0.075
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.905
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS9
|
0.471
|
0.665
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.423
|
0.22
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
EEF1A1
|
0.42
|
-0.536
|
-0.552
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.482
|
0.352
|
0.334
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS15
|
0.383
|
-0.043
|
-0.155
|
0.519
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.524
|
0.946
|
0.804
|
0.37
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS23
|
0.194
|
0.591
|
0.528
|
-0.129
|
-0.48
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.754
|
0.294
|
0.36
|
0.836
|
0.413
|
-
|
-
|
-
|
-
|
-
|
UXT
|
-0.247
|
0.571
|
0.324
|
-0.569
|
0.386
|
-0.318
|
-
|
-
|
-
|
-
|
p-value
|
0.689
|
0.315
|
0.594
|
0.317
|
0.521
|
0.602
|
-
|
-
|
-
|
-
|
RPL4
|
-0.471
|
0.569
|
0.366
|
-0.845
|
-0.029
|
-0.204
|
0.908
|
-
|
-
|
-
|
p-value
|
0.423
|
0.317
|
0.545
|
0.071
|
0.963
|
0.741
|
0.033
|
-
|
-
|
-
|
B2M
|
0.555
|
0.222
|
0.734
|
-0.382
|
0.28
|
-0.165
|
0.516
|
0.427
|
-
|
-
|
p-value
|
0.332
|
0.72
|
0.158
|
0.526
|
0.648
|
0.79
|
0.373
|
0.473
|
-
|
-
|
HPRT1
|
0.096
|
0.253
|
0.315
|
-0.347
|
0.592
|
-0.549
|
0.884
|
0.706
|
0.755
|
-
|
p-value
|
0.878
|
0.681
|
0.606
|
0.567
|
0.293
|
0.338
|
0.047
|
0.183
|
0.14
|
-
|
|
BestKeeper vs.
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
coeff. of corr. [r]
|
0.372
|
0.677
|
0.751
|
0.001
|
0.446
|
0.055
|
0.774
|
0.599
|
0.809
|
0.797
|
p-value
|
0.538
|
0.21
|
0.144
|
0.51
|
0.451
|
0.93
|
0.124
|
0.285
|
0.097
|
0.107
|
Zanskar Horses (ZAP)
The M values calculated using geNorm analysis for all the RGs in Zanskar ponies are shown in Table 3. Except, B2M and GAPDH RGs, the M values for all other RGs were within the acceptable limit of <1.5. The M value for all the RGs in ZAP ranged from 0.135 to 1.721. The ranking order of RGs was as follows; RPS9=RPL4 > RPS23> EEF1A1> UXT> RPS15> HPRT1> ACTB> B2M> GAPDH (Fig. 3P). The two most stable RGs with lowest M value were RPS9 and RPL4 (0.135) while GAPDH and B2M were the least stable RGs with M value of 1.712 and 1.394., respectively. Further, the V values for V2/3, V3/4, V4/5 and V5/6 were within the threshold limit of 0.15. Based on geNorm analysis, the geometric mean of RPS9, RPL4 and RPS23 RGs is likely to provide accurate normalization of gene expression data in ZAP (Fig. 3Q).
In Normfinder analysis ranking of genes in high altitude ZAP from most stable to least stable was as follows: EEF1A1 (0.631), UXT (0.684), RPS9 (0.749), RPS23 (0.759), RPL4 (0.763), RPS15 (1.022), ACTB (1.36), HPRT1 (1.727), B2M (1.945), GAPDH (2.795) (Fig. 3R)
From BestKeeper algorthim, UXT gene revealed minimum SD value of 0.341 with smallest variation, followed by RPS23, RPS9, RPL4, EEF1A1, RPS15, ACTB, HPRT1, B2M and GAPDH with the SD value 0.434, 0.465, 0.469, 0.498, 0.816, 1.211, 1.226, 1.914, 2.099 respectively (Table 14). The best correlation between RGs and BestKeeper was observed for B2M (r=0.947) and ACTB (r=0.662) (Table 15). The high correlation values for these genes indicated their reliability as RGs.
Table 14 Analysis of parameters based quantitative cycling points (CP) for 10 candidate RGs in ZAP
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
n
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
geo Mean [CP]
|
21.69
|
17.49
|
20.99
|
19.82
|
35.03
|
17.55
|
27.02
|
18.71
|
17.65
|
24.91
|
AR Mean [CP]
|
21.81
|
17.54
|
21
|
19.83
|
35.04
|
17.56
|
27.02
|
18.72
|
17.75
|
24.96
|
min [CP]
|
19.69
|
16.4
|
19.84
|
18.81
|
33.61
|
16.47
|
26.17
|
17.55
|
16.06
|
22.06
|
max [CP]
|
25.66
|
19.94
|
21.45
|
20.5
|
36.06
|
18.13
|
27.41
|
19.21
|
20.49
|
26.81
|
std dev [+/- CP]
|
2.1
|
1.21
|
0.46
|
0.5
|
0.82
|
0.43
|
0.34
|
0.47
|
1.91
|
1.23
|
CV [% CP]
|
9.63
|
6.91
|
2.21
|
2.51
|
2.33
|
2.47
|
1.26
|
2.5
|
10.78
|
4.91
|
min [x-fold]
|
-4
|
-2.12
|
-2.22
|
-2.02
|
-2.67
|
-2.11
|
-1.8
|
-2.24
|
-3
|
-7.21
|
max [x-fold]
|
15.68
|
5.48
|
1.37
|
1.6
|
2.05
|
1.5
|
1.31
|
1.41
|
7.18
|
3.73
|
std dev [+/- x-fold]
|
4.28
|
2.32
|
1.38
|
1.41
|
1.76
|
1.35
|
1.27
|
1.38
|
3.77
|
2.34
|
Table 15 Analysis of repeated pair-wise correlation amongst genes in ZAP with BestKeeper index
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
ACTB
|
0.715
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.175
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS9
|
-0.762
|
-0.798
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.134
|
0.106
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
EEF1A1
|
-0.586
|
-0.615
|
0.913
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.299
|
0.27
|
0.031
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS15
|
-0.734
|
-0.469
|
0.826
|
0.647
|
-
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.158
|
0.425
|
0.085
|
0.238
|
-
|
-
|
-
|
-
|
-
|
-
|
RPS23
|
-0.628
|
-0.82
|
0.976
|
0.906
|
0.728
|
-
|
-
|
-
|
-
|
-
|
p-value
|
0.257
|
0.089
|
0.004
|
0.034
|
0.164
|
-
|
-
|
-
|
-
|
-
|
UXT
|
-0.881
|
-0.922
|
0.917
|
0.8
|
0.656
|
0.876
|
-
|
-
|
-
|
-
|
p-value
|
0.049
|
0.026
|
0.028
|
0.104
|
0.229
|
0.051
|
-
|
-
|
-
|
-
|
RPL4
|
-0.685
|
-0.777
|
0.981
|
0.966
|
0.723
|
0.979
|
0.899
|
-
|
-
|
-
|
p-value
|
0.202
|
0.122
|
0.003
|
0.008
|
0.167
|
0.004
|
0.038
|
-
|
-
|
-
|
B2M
|
0.434
|
0.854
|
-0.388
|
-0.128
|
-0.095
|
-0.446
|
-0.61
|
-0.347
|
-
|
-
|
p-value
|
0.466
|
0.065
|
0.519
|
0.837
|
0.879
|
0.452
|
0.275
|
0.568
|
-
|
-
|
HPRT1
|
-0.503
|
0.244
|
0.046
|
0.019
|
0.415
|
-0.169
|
0.073
|
-0.039
|
0.433
|
-
|
p-value
|
0.387
|
0.693
|
0.941
|
0.975
|
0.488
|
0.786
|
0.907
|
0.95
|
0.467
|
-
|
BestKeeper vs.
|
GAPDH
|
ACTB
|
RPS9
|
EEF1A1
|
RPS15
|
RPS23
|
UXT
|
RPL4
|
B2M
|
HPRT1
|
coeff. of corr. [r]
|
0.257
|
0.662
|
0.001
|
0.159
|
0.182
|
0.001
|
0.001
|
0.001
|
0.947
|
0.427
|
p-value
|
0.676
|
0.224
|
0.894
|
0.799
|
0.77
|
0.821
|
0.533
|
0.939
|
0.014
|
0.473
|
In RefFinder analysis, RPS9, RPL4 and UXT were overall most stable while GAPDH, B2M and HPRT1 were the least stable. Based on all the methods; geNorm Normfinder, BestKeeper and RefFinder RPS9, RPL4 and UXT were observed to be most stable RGs in ZAP.