Abstract
Relative real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a well-established method for the precise quantification of gene expression. For accurate relative real-time RT-qPCR analysis, validation of the expression of an appropriate reference gene is required. In this study, the expression of six commonly used reference genes, namely 40S ribosomal protein (40S), actin (ACT), cyclophilin C (CYCC), EF-1 alpha (EF1), TATA box binding protein (TBP) and polyubiquitin (UBI) was investigated in leaf and root samples of cassava obtained at 6, 9 and 12 months after planting (MAP). A transcript stability analysis was undertaken in two different varieties of cassava, namely Huay Bong 60 which has high cyanogenic potential (CN) and Hanatee which has low CN. The results reveal that TBP was the most stable reference gene for expression studies. This information was applied to an analysis of linamarase and α-hydroxynitrile lyase gene expression in samples from six low and six high CN cassava plants collected at 6, 9 and 12 MAP. The results indicate that at 6 MAP, the linamarase transcript from leaf of the high CN group was significantly increased, and the α-hydroxynitrile lyase transcript was significantly increased at 12 MAP. Key words: Cassava, housekeeping gene, hydroxynitrile lyase, linamarase, real-time polymerase chain reaction (PCR).
Highlights
Accurate quantification of gene expression is important in a number of experimental situations, such as in determining the alteration in expression levels occurring at a defined biological stages and in detecting the response of genes to various stimuli (Fraga et al, 2008)
In order to determine the most stable reference genes, expression levels of transcripts from these six reference genes were determined in Huay Bong 60 and Hanatee, which are significantly different in cyanogenic potential (CN) (Whankaew et al, 2011) at 6, 9 and 12 month after planting (MAP)
TATA box binding protein (TBP) was the most stable reference gene transcript evaluated with a stability value of 0.01 across samples
Summary
Accurate quantification of gene expression is important in a number of experimental situations, such as in determining the alteration in expression levels occurring at a defined biological stages and in detecting the response of genes to various stimuli (Fraga et al, 2008). Since the stability of reference gene for expression study of genes affecting cyanogenic potential (CN) has not been identified elsewhere, this research sought to validate potential reference transcripts across growing stages and tissues of two varieties of cassava with different endogenous CN in order to provide a transcript suitable for the accurate normalization of realtime quantitative RT-PCR data. The study utilized the most stable transcript to investigate the expression of LNM and HNL genes at 3 growing stages of cassava in vivo. Both root and leaf tissues were used and six plants each of low and high CN were analyzed, in order to understand more about transcription pattern at each stage and tissue systematically
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