We tested the amplification efficiency before using housekeeping genes. Referring to the previous study of Arabidopsis housekeeping genes (Dheda, 2004; Czechowski, 2005; Hou, 2018), we selected Atactin1 (F: AGGCACCTCTTAACCCTAAAGC, R: GGACAACGGAATCTCTCAGC), Atactin2 (F: TTGTGCTGGATTCTGGTGATGG, R: CCGCTCTCTGCTGTTGTGGTG) and Atactin8 (F: GAATTACCCGACGGACA, R: ACGGTCTGCAATACCT) were tested for efficiency, and we found that the best result was Atactin2. The best housekeeping gene for O. fragrans is OfACT, which has been reported in a previous study (Zhang, 2015). Dheda, K.; Huggett, J.F. ; Bustin, S.A. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques. 2004, 37: 112-119. https://doi.org/10.2144/04371RR03 Czechowski, T.; Stitt, M.; Altmann, T. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 2005, 139: 5-17. https://doi.org/10.1104/pp.105.063743 Hou, H.; Jia, H.; Yan, Q.; Wang, X. Overexpression of a SBP-Box Gene (VpSBP16) from Chinese Wild Vitis Species in Arabidopsis Improves Salinity and Drought Stress Tolerance. Int. J. Mol. Sci. 2018, 19, 940. https://doi.org/10.3390/ijms19040940 Zhang, C.; Fu, J.X. ; Wang, Y.G. ; Bao, Z.Y. ; Zhao, H.B. Identification of Suitable Reference Genes for Gene Expression Normalization in the Quantitative Real-Time PCR Analysis of Sweet Osmanthus (Osmanthus fragrans Lour.). Plos one. 2015, 10,1-17. https://doi.org/10.1371/journal.pone.0136355 Point 3: Line 124: generat instead of generate.
Auhtors report that Actin was used as housekeeping gene. Do the authors have performed an efficiency test before using it? Do they tried to use otherHK genes?