In our study, 7 candidate reference genes that are most frequently used in RT-qPCR investigations were assessed (Table 1 ). The primers were designed based on the PrimerQuest Tool1 , according to the sequences obtained from our recently sequenced transcriptomes for H. vigintioctopunctata (unpublished data).
The PCR reaction system and parameters were used according to our previous study (Yang et al., 2014 (link)). Amplicons of the expected lengths were purified using the TIANgel Midi Purification Kit (Tiangen, China), and subcloned into the pClone007 Blunt vector before transformation into Escherichia coli DH5α competent cells (Tsingke, China) for sequencing by Tsingke company. The reference genes were confirmed using sequence analysis.
The PCR reaction system and parameters were used according to our previous study (Yang et al., 2014 (link)). Amplicons of the expected lengths were purified using the TIANgel Midi Purification Kit (Tiangen, China), and subcloned into the pClone007 Blunt vector before transformation into Escherichia coli DH5α competent cells (Tsingke, China) for sequencing by Tsingke company. The reference genes were confirmed using sequence analysis.
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