Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane

Table 2

Parameters derived from RT-qPCR data analysis^∗, including PCR amplification efficiency (𝐸) established by calibration curves for each quantified target.

Gene	Tm (°C)	Product size (bp)	Average Cq	𝐸 (%)	NTC (Cq)	Correlation coefficient (𝑅)	Slope	𝑌 intercept
𝛼TUB	75.9	104	27.48	99.53	35.82	0.998	−3.33	37.34
GAPDH	81.8	100	23.69	100.89	N.D.	0.984	−3.30	41.40
H1	78.0	57	24.76	97.41	33.59	0.999	−3.39	37.92
SAMDC	79.0	60	22.74	99.66	36.62	0.992	−3.33	34.36
UBQ	81.2	153	24.64	98.34	N.D.	0.999	−3.36	37.86
25S rRNA	82.9	108	14.00	99.82	N.D.	0.999	−3.33	35.54
PFP𝛼1	83.6	151	21.92	92.99	32.90	0.988	−3.50	40.44
AS	79.8	112	24.94	105.05	N.D.	0.990	−3.21	40.26
PIP1-1	84.6	134	24.47	95.55	N.D.	0.995	−3.43	37.50
ACC oxidase	82.2	152	30.21	93.52	N.D.	0.990	−3.49	44.65

∗Based on MIQE Guidelines [26]. Tm: melting temperature (°C); N.D.: not detected; NTC: no template control; 𝛼TUB: alpha-tubulin; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; H1: histone H1; UBQ: ubiquitin; SAMDC: S-adenosylmethionine decarboxylase; 25S rRNA: 25S ribosomal RNA; PFP𝛼1: pyrophosphate fructose-6-phosphate 1-phosphotransferase alpha subunit (EC 2.7.1.90); AS: glutamine-dependent asparagine synthetase (EC 6.3.5.4); PIP1-1: plasma membrane intrinsic protein; ACC oxidase: 1-aminocyclopropane-1-carboxylate oxidase (EC 1.14.17.4).