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Fig. 3 | BMC Research Notes

Fig. 3

From: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

Fig. 3

Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1, b brm, c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS-RNAi constructs with Act-Gal4. Expression levels were normalized to the reference genes, eIF2Bγ and βCOP. Shown here, are relative expression values compared to the UAS-mCherry-RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Error bars indicate the standard error of the mean

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