Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT‐PCR

Abstract

Gene expression quantification at mRNA level is very important for post‐genomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real‐time polymerase chain reaction (qRT‐PCR), require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome‐wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT‐PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 (represented by sw22934, swl4876, and swl3956) were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin (sw22671), the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.

Stable reference genes for the measurement of transcript abundance during larval caste development in the honeybee

“…The genes in Table I had been previously investigated as reference genes in honeybees, silk moth (Bombyx mori) (Wang et al 2008) or springtails (Folsomia candida / Orchesella cincta) (de Boer et al 2009). Table II shows the details the genes identified as having stable expression throughout larval development between queens and workers based on microarray analysis.…”

Selection of reference genes for analysis of stress-responsive genes after challenge with viruses and temperature changes in the silkworm Bombyx mori

“…TIFs have been identified as housekeeping genes and are widely used as RGs for gene expression analysis (Eisenberg and Levanon 2003;Nicot et al 2005;Wang et al 2008;Zhou et al 2013). Wang et al (2008) reported that TIF-4A is highly stable in silkworms; therefore, we did not perform stability analysis in the present study.…”

“…qPCR is a common tool for analysis of the expression levels of stressresponsive genes, and identification of appropriate RGs is critical for data normalization in qPCR (Huggett et al 2005;Wang et al 2008;Velada et al 2014). Therefore, in this study, we summarized the RGs used in the previous studies of silkworms and identified appropriate RGs that should be used in the context of various commonly applied treatments.…”

“…Some interesting candidate genes have been used for further verification by quantitative polymerase chain reaction (qPCR) (Bao et al 2009;Xue et al 2012;Gao et al 2014b;Li et al 2014). However, reliable quantification of gene expression by qPCR may be affected by variations in initial sample quantity, RNA integrity, and reverse transcription efficiency (Huggett et al 2005;Wang et al 2008;Velada et al 2014). Therefore, normalization is necessary to correct these variations.…”

Bombyx mori nucleopolyhedrovirus lef8 gene: effects of deletion and implications for gene transduction applications

“…The results were processed through the LinRegPCR software (Ruijter et al, 2009) and were normalized to the respective values of the cellular reference gene, Transcription Initiation Factor 3, subunit 4 (TIF; Wang et al, 2008). TIF remained reliable up until the 71 h time point, and results concerning transcription are thus presented up to that point.…”