Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae

Figueiredo, Andreia; Loureiro, Andreia; Batista, Dora; Monteiro, Filipa; Várzea, Vítor; Pais, Maria Salomé; Gichuru, Elijah K; Silva, Maria Céu

doi:10.1186/1756-0500-6-388

Table 1 Candidate reference genes and target genes primer sequences, amplicon length and qPCR analysis

From: Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae

Gene (coffee	Reference	Primer sequence	Amplicon length (bp)	Ta (°C)	Tm (°C)	PCR efficiency (%)	Regression coefficient (R²)	Average Cq value
source gene)
VATP16 (GT00156.1)*	Gamm et al. [29]	Fw: ATAGAGAGAGAGCCCCCAATTC	150	60	78.63	Discarded (unspecific amplification, primer dimer)
VATP16 (GT00156.1)*		Rev: CCGAGGAAGCCAAAAAAAG	150	60	78.63
UQCC (isotig09120)		Fw: CCTCGGGCTTCATCTCTACTC	156	60	77.21
UQCC (isotig09120)		Rev: TCTCCCCAGCATTTTTTGTC	156	60	77.21
SAND (isotig05620)	Selim et al. [30]	Fw: GATTTGTCTACGAACCCTGCTT	116	60	78.70
SAND (isotig05620)	Selim et al. [30]	Rev: GGACCACCAACACCAATAAAC	116	60	78.70
UBQ9 (AF29708)*	Ramiro et al. [7]	Fw: AACATTGAGGGTGGTTCTGTTC	79	60	76.16	92	0.987	24.5 ± 0.95
UBQ9 (AF29708)*	Ramiro et al. [7]	Rev: GCAGAAAACCAACTAAGACCTAACAA	79	60	76.16	92	0.987	24.5 ± 0.95
S24 (SNG-U349723)^a	Cruz et al. [28]	Fw: GCCCAAATATCGGCTTATCA	92	60	76.60	91	0.996	20.61 ± 1.04
S24 (SNG-U349723)^a	Cruz et al. [28]	Rev: TCTTCTTGGCCCTGTTCTTC	92	60	76.60	91	0.996	20.61 ± 1.04
IDE (isotig10635)	Borges et al. [12]	Fw: TGATCTAAGCTGGTGGAAAGC	91	55	76.28	92	0.994	24.46 ± 1.30
IDE (isotig10635)		Rev: TCAGGTGCATCAGGATGATT	91	55	76.28	92	0.994	24.46 ± 1.30
β- Tub9 (isotig08544)		Fw: ACCCTCCAGCAAACTGATGA	100	55	77.27	96	0.996	19.40 ± 0.82
β- Tub9 (isotig08544)		Rev: AGGATGCCACTGCTGATGAT	100	55	77.27	96	0.996	19.40 ± 0.82
14-3-3 (SGN-U356404)^a	Barsalobres-Cavallari et al. [18]	Fw: TGTGCTCTTTAGCTTCCAAACG	75	60	73.33	103	0.999	22.76 ± 1.47
14-3-3 (SGN-U356404)^a		Rev: CTTCACGAGACATATTGTCTTACTCAAA	75	60	73.33	103	0.999	22.76 ± 1.47
GADPH (SGN-U347734)^a		Fw: TTGAAGGGCGGTGCAAA	59	60	75.73	96	0.998	21.42 ± 1.96
GADPH (SGN-U347734)^a		Rev: AACATGGGTGCATCCTTGCT	59	60	75.73	96	0.998	21.42 ± 1.96
RPL7 (SGN-U351477)^a	Barsalobres-Cavallari et al. [18]	Fw: CATTCGAGGTATCAATGCTATGCA	66	60	76.48	89	0.999	23.76 ± 1.28
RPL7 (SGN-U351477)^a	Barsalobres-Cavallari et al. [18]	Rev: TGTCTCAGGCGCAGAAGCT	66	60	76.48	89	0.999	23.76 ± 1.28
Genes of interest (GOIs)
PR10 (CF589103)*	Ramiro et al. [7]	Fw: GCCACCATCCTTGAAGAGAA	151	55	80.17	99	0.999	19.35 ± 2.28
PR10 (CF589103)*		Rev: CAACTCTCTGCTTGGCAGTCT	151	55	80.17	99	0.999	19.35 ± 2.28
RLK (CF589181)*		Fw: ATGGGAGAAAAGAATGGCAGAAG	189	55	81.15	91	0.998	24.81 ± 2.02
RLK (CF589181)*		Rev: GGCCAATTACAGTTTGAAAACACC	189	55	81.15	91	0.998	24.81 ± 2.02

^aUnigene accession number according to the SOL Genomics Network.
Ta, annealing temperature; Tm, melting temperature; SD, standard deviation. * NCBI accession number or TC TIGR number.

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ISSN: 1756-0500

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