Identification of reference genes for tissue-specific gene expressionin Panax notoginseng using quantitative real-time PCR

Qiong Wu*·Xinye Ma·Kefeng Zhang·Xuehua Feng

*The correspondence author.E-mail address:

Supplementary Table 1. The RT-PCR primer sequences, amplicon size (bp), length of cDNA sequence and accession no. of the candidate genes in P. notoginseng

Primer name / Gene name / Primer sequence (5′-3′)a / Amplicon size (bp) / Accession no. / Length (bp) of cDNA sequence
26S1-Q / 26 S ribosomal RNA / F: CGGTCTCTCGCCAGTATTTAGC
R: GGGAATGCAGCCCCAATC / 61 / EC599968 / 109
26S2-Q / 26 S ribosomal RNA / F: CAGTATTTAGCCTTGGACGGAATT
R: CGGGTTGTTTGGGAATGC / 60 / EC599969 / 112
ACT1-Q / actin / F: TCCGTTGCCCAGAAGTCCTA
R: TCCCTGCAGCTTCCATTCC / 57 / KF815705 / 1770
ACT2-Q / actin / F: GCCGATCTTTCCCTGTATGC
R: CATCACCAGAATCCAGCACAA / 61 / KF815706 / 1189
CYC-Q / Cyclophilin / F: GGGCCCGGAACCAATG
R: CAGCCACTCCGTCTTTGCA / 57 / KF815707 / 977
EF-Q / Elongation factor / F: CCTTGGAGTTGGATGCAACA
R: CCACTATATCGTGCAACGAGTCA / 62 / KF815708 / 2221
ETIF1-Q / Eukaryotic translation initiation Factor / F: GGCTTCCCACCGATGAAAAT
R: TCCCTTCAGCAAACCCATCT / 57 / KF815709 / 835
ETIF2-Q / Eukaryotic translation initiation Factor / F: GAGAAGTGTGGCTCTGGAAGGT
R: GGCCAACACGAGCAACAAA / 64 / KF815710 / 2220
GAPDH-1 / Glyceraldehyde-3-phosphate dehydrogenase / F: GCCAAGGCTGTTGGTAAAGTG
R: GGAAGGCCATTCCAGTCAATT / 61 / DQ186631 / 627
GAPDH2-Q / Glyceraldehyde-3-phosphate dehydrogenase / F: GGGCTGCCAAGGCTGTT
R: AGGCCATTCCAGTCAATTTCC / 63 / KF815711 / 2368
TUB1-Q / tubulin / F: GTTGGCGGAGGTGATGATG
R: CTTTCCTGCGCCAGTTTCA / 57 / KF815712 / 1738
TUB2-Q / tubulin / F: CGGAGGAAAGTTTTGGGAAGA
R: CCCGGTGGCGTCGAT / 55 / KF815713 / 3861
UBI1-Q / Ubiquitin / F:GTACCACAAGACCATAGTCACCAGAT
R: TCGTCTCCGTGGTGGTTTC / 64 / EC600045 / 616
UBI2-Q / Ubiquitin / F: TCCAAGACAAGGAGGGCATT
R: GCTTTCCCGCGAAGATGAG / 57 / KF815714 / 639
DDS-Q / Dammarenediol synthase / F:CAAACTGCGAAAAAGAGGTCTAAAA
R:TGGTCTCGGTGGCACCATA / 65 / KC953035 / 2310

aThe capital letter F and R is the abbreviation of forward and reverse, respectively.

Supplementary Table 2. The slope, Y-Inter, R2and amplification efficiency (Eff %) for candidate genes

No. / Target / Slope / Y-Inter / R2 / Eff%a
1 / 26S-2 / -3.326 / 30.615 / 0.984 / 99.851
2 / ACT-2 / -3.284 / 46.164 / 0.981 / 103.172
3 / CYC / -3.873 / 43.802 / 0.992 / 81.214
4 / ETIF-2 / -3.535 / 45.248 / 0.962 / 91.809
5 / GAPDH / -3.608 / 41.56 / 0.963 / 89.312
6 / TUB / -3.444 / 42.55 / 0.971 / 95.165
7 / DDS / -3.511 / 44.86 / 0.994 / 92.687

a The amplification efficiency was then calculated based on the slope of the regression line in the standard curve for each gene using the following equation: Efficiency%=(10(-1/slope)-1)×100%.

SupplementaryFigure 1. Standard curves of six reference genes and DDS gene were generated from qRT-PCR analyses.The symbol A to G represents 26S-2, ACT-2, CYC, ETIF-2, GAPDH, TUB, and DDS, respectively.

Supplementary Figure 2. Average expression stability (M) of six selected reference genes measured by geNorm values.

Supplementary Figure 3. Optimal number of reference genes for normalization determined by the pairwise variation V. Vn/Vn+1 is the pairwise variation between normalization factors of n and n+1 genes.

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