Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data

Table 1

Primers used for RT-qPCR normalization.

Gene abbreviation	Gene name	Primer sequence (5′–3′)	Amplicon length (bp)	Primers Tm (°C)	E (%)	R^2
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	For: GCTGAGATTCTTGATGGGGAG	101	58.7/58.5	97.90	0.998
		Rev: CTTGTGGTAGGATTTACTATGGCA
eIF	Eukaryotic translation initiation factor	For: TCAGTTTACTCGTGGGACATCG; Rev: GGTTCACATACGCAGCCTCG	181	60.1/61.3	93.58	0.992
EF-1α	Elongation factor 1-alpha	For: GAGGTTTTGAGGCTGGCATT	117	59.2/59.9	102.78	0.995
		Rev: GGGTGGTGGCATCCATCTT
SAND	SAND family protein	For: GCAACTGAATCCACAGAGCG	259	58.9/59.4	94.18	0.998
		Rev: CCCAGCAAAGCAGAACATAATC
PP2A	Serine/threonine-protein phosphatase 2A	For: TTGGTTCTTCGCAGTTGATTG	208	58.8/59.0	103.91	0.995
		Rev: AGCCCGCTATTGACCTTGTT
hnRNP	Heterogeneous nuclear ribonucleoprotein	For: GCAAAGGCAGCACTGTCAAG	187	59.2/61.1	99.36	0.996
		Rev: CGGTAATGTTCCCGTATTTCTCA
UBCE	Ubiquitin-conjugating enzyme	For: CCCAATCCGTCTGACCCTT	203	59.3/61.7	108.08	0.99
		Rev: GCTTTTCCAGCAACTTCCTCG
RPL5	60S ribosomal protein L5	For: TGGTGGGCTTGACATTCCT	150	58.2/61.5	93.62	0.994
		Rev: CGCTCTGGCTCATCTTCTTCC
PTBP2	Polypyrimidine tract-binding protein homolog 2	For: GGAGCAAATCGTAACCAAGCA	115	60.4/59.0	93.36	0.999
		Rev: AGACAGTTTTCCCTCGCACC
TBP	TATA binding protein	For: GGGCTTGCTTACTCTCACGG	146	59.9/58.4	101.57	0.996
		Rev: TCCTCCCTAACCTTTGCTCC
TUB	Beta-1 tubulin	For: CTCTTCAAGTAGGTCAATGTGGG	116	58.3/58.4	98.83	0.995
		Rev: CCCTCCCTGAGTAGCGAAGT
ACT2	Actin-related protein 2	For: ATTGTCGTTGGAGATGCTTGC	149	60.1/61.7	102.79	0.995
		Rev: CATTCCGTTGGGTCTAACTTCAG
MDH	Malate dehydrogenase	For: ATCCAGTCAGATGCGTCGG	119	58.7/59.5	95.76	0.998
		Rev: GCTTTCTCTTCATACTCGGTCAAC
PEPC	Phosphoenolpyruvate carboxykinase	For: TTTGGTGTCCTTCCCCCTG	106	60.2/60.8	94.66	0.999
		Rev: CCTTGATTCCATCCTCGGTTC
F-box	F-box protein	For: AGTTCCACCGCTACCTGTCC	129	58.9/58.8	99.03	0.996
		Rev: ATTCGCCTTCAACGCCAA
OSBL	o-Succinylbenzoate-CoA ligase	For: TGCTGGCTACACTGAGGATGA	143	58.9/60.3	95.27	0.999
		Rev: CCTTGACCGCTGCTTGAACT
ICS	Isochorismate synthase	For: CATCCCTTCATCCAACTCCAG	218	59.2/58.5	98.46	0.998
		Rev: GCTTCCTTCTACCACGCCA
IPPI	Isopentenyl-diphosphate delta-isomerase	For: AACCGAGACGAGTTGAGGGA	152	59.3/58.0	93.75	0.996
		Rev: ATGTCAATAGCATCAGTCAGGGT
HMGR	3-Hydroxy-3-methylglutaryl-coenzyme A reductase	For: TTGAGGTCGGGACAGTAGGTG	116	59.8/58.6	93.79	0.998
		Rev: CAGCAGCCGAGCATTTGA
DXS	1-Deoxy-D-xylulose-5-phosphate synthase	For: TTCTCTGCCTACGGCTACTCTT	119	58.2/58.8	94.68	0.996
		Rev: AACTTTTCGCTGCCTCGC
AACT	Acetyl-CoA acetyltransferase	For: TTGAGGTCGGGACAGTAGGTG	249	60.0/59.4	96.41	0.994
		Rev: CAGCAGCCGAGCATTTGA

bp, Tm, E, and R² indicate base pair, melting temperature, PCR efficiency, and correlation coefficient, respectively. Standard curves of 15 candidate reference genes and six target genes are shown in Additional Figure S1.