Transcript Expression Analysis of JH/IIS Genes

We used the same dissection and total RNA extraction methods described for miRNA RT-qPCR and the same total RNA for both miRNA and mRNA RT-qPCR. cDNA was synthesized using M-MuLV Reverse Transcriptase (NEB, Ispwich, MA, USA) and RNase Inhibitor, Human Placenta (NEB). An exogenous RNA (Root Cap Protein 1 from Arabidopsis thaliana) was added at the cDNA step to assess cDNA synthesis efficiency. mRNA expression was measured for four genes involved in JH/IIS: insulin-like peptide receptor (InR-1) (Ament et al., 2008 (link)), Krüppel homolog 1 (Kr-h1) (Grozinger et al., 2003 (link)), ultraspiracle (USP) (Ament et al., 2012a (link)) and vitellogenin (Vg) (Pinto et al., 2000 (link)). Primer sequences can be found in Table S1. qPCR measurements were performed in triplicate and expression was normalized to the geometric mean of the expression of endogenous genes Rp49, S8 and GapDH.