Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR
Table 1
Primers for real-time RT-PCR and their characteristics.
(a)
Gene
Primer sequences (forward/reverse)
Length (bp)
ACTα
TTGTTAGCAACTGGGATGATATGG/CAGGGTGTTCTTCAGGAGCAA
84
ARPT
GCTGTTCAAGCTGGAGAACGT/TTCCGCCAGTTGCAATGA
58
CYP
TGACTTCACCGCCGGAAA/GGCGAACTTGGCTCCATAGA
58
EF1
CCGAGCGTGAACGTGGTAT/TAGTACTTGGTGGTTTCGAATTTCC
64
EIF1
CCTCCGTATGCTTGGAAACG/TCTTGGTTCCGTCGATGCA
56
GAPD
GGCATTGTTGAGGGTCTTATGAC/TGGTCCATCAACAGTCTTCTGAGT
69
rbcL
CGGTGTGGACCGATGGA/TGGTAGCACCGCCCTTTG
55
TUBα
TCAGGAAGCTTGCAGACAACTG/AACAGCGTGGAAAACCAGAAA
59
TUBβ
GGGCCAAAGGGCATTACACT/CGACATCGAGAACAGCATCAA
57
UBQ
CCTGACCAGCAGAGGTTGATC/TCCGCCAGAGTCCTTCCAT
62
IPPI
AACGATCAGGGACAAAGGTAACA/GGATGGCTGCAGCATGTG
61
FPPS1
AAGGAGCTCAACTCACTGAGAGC/AATGCACCAACCCAACACAG
59
(b)
Tm (°C)
Ct value
Standard curve
Average
SD
Range
CV (%)
Slope
PCR efficiency (%)
76.5
22.738
1.192
5.489
5.243
0.927
99.67
77.2
19.216
0.635
2.063
3.302
0.953
104.23
79.1
28.035
0.750
3.696
2.673
0.932
116.50
76.0
16.407
0.983
3.994
5.991
0.955
100.62
79.3
19.232
1.040
3.971
5.408
0.941
108.04
78.0
17.938
0.853
3.788
4.758
0.955
75.07
79.0
13.330
0.905
3.347
6.792
0.941
115.95
78.6
19.521
0.988
3.777
5.060
0.970
105.17
77.0
21.242
1.426
4.973
6.714
0.901
124.50
79.5
15.906
0.819
2.664
5.148
0.941
119.49
79.3
23.070
1.740
5.706
7.541
0.966
96.48
77.6
20.292
1.309
4.335
6.449
0.981
100.38
Data are based on analyses of absolute Ct values from all tested samples and standard curve of each target (IPPI, FPPS1) and candidate housekeeping gene. Tm: melting temperature of real-time RT-PCR product; SD: standard deviation; Range: difference between the maximum and minimum Ct values; CV: coefficient of variation; Slope: slope of standard curve; : correlation coefficient of standard curve; PCR efficiency = (00%.
