M mulv reverse transcriptase

Total RNA from cell cultures was isolated using Trizol (Invitrogen) and then treated with DNase I before reverse transcription processing to remove genomic DNA contamination. The reverse transcription was performed according to the manufacturer’s protocol of M-MULV reverse transcriptase (New England BioLab, USA). Briefly, a total of 2 µg RNA from each sample was incubated in a 20 µL reaction volume containing 10× buffer for M-MULV reverse transcriptase (New England BioLab, USA), 20 U RNAse inhibitor (New England BioLab, USA), 1 mM dNTPs, 0.5 µg/µL random primers (Promega), and 200 U M-MULV reverse transcriptase (New England BioLab, USA) for 5 min at 37°C followed by 60 min at 42°C and 10 min at 70°C. Parallel reactions were performed in the absence of reverse transcriptase to control for the presence of contaminant DNA.

RT-PCR and real-time qPCR were performed as previously described 20 (link). Briefly total RNA from cell cultures was isolated using TRIzol™ (Invitrogen, USA) and then incubated with DNase I (Invitrogen) before RT. For RT, 2 µg RNA/sample was incubated in a 20 µL reaction volume containing 10× buffer for M-MuLV reverse transcriptase (New England BioLab, USA), 20U RNAse inhibitor (New England BioLab, USA), 1mM dNTPs, 0.5 µg/µL random primers (Promega, USA), and 200 U M-MuLV reverse transcriptase (New England BioLab, USA) at 37 °C for 5 min, at 42 °C for 60 min and at 70 °C for 10 min. For real-time qPCR, reactions were prepared with Hot FIREPol® DNA polymerase (Solis Biodyne, Estonia) to a final volume of 20 µL containing 2 µL cDNA diluted 1:1 and 500 nM primer (Table S2), and carried out in an Mx3000P QPCR System (Agilent Technologies, USA). Thermal cycling conditions were: 10 min denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 20 s, and extension at 72 °C for 20 s. The relative changes in gene expression were calculated with the relative quantification method (2^-ΔΔCt) and normalized according to the expression in basal conditions.

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.