Retro transcriptase ssii

Total Arabidopsis RNA and miRNA were isolated using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and MIRVANA (Ambion, Austin, TX, USA), respectively, was quantified by UV spectrophotometry and its integrity was visually assessed on ethidium bromide-stained agarose gels. After treatment with DNase I Amp Grade (Invitrogen, Waltham, MA, USA), cDNA was generated by retro-transcriptase SSII (Invitrogen, Waltham, MA, USA) as previously described [76 (link),80 (link)]. Real-time quantitative PCR (RT-qPCR) or stem-loop RT-qPCR was carried out with SYBRGreen qPCR Super-Mix-UDG with ROX (Invitrogen, Waltham, MA, USA), with the specific primers detailed in Supplementary Tables S2 and S3, respectively, in a CFX96 Touch™ Real Time PCR Detection System (BioRad, Hercules, CA, USA), with one cycle of 95 °C for 2 min and 40 cycles consisting of 95 °C for 30 s and 60 °C for 30 s or one cycle of 95 °C for 3 min or 60 cycles consisting of 95 °C for 20 s, 53 °C for 90 s and 50 °C for 30 s, respectively. Expression values were normalized to UBQ10 or 18S genes, respectively, using the 2^−ddCt method [81 (link)].

Total Arabidopsis RNA was isolated using the RNeasy Plant Mini Kit (Qiagen), was quantified by UV spectrophotometry and its integrity was visually assessed on ethidium bromide-stained agarose gels. After treatment with Dnase I Amp Grade (Invitrogen), cDNA was generated by retro-transcriptase SSII (Invitrogen) as previously described (Andrés-Colás et al., 2006 (link)). Real-time quantitative PCR (RT-qPCR) was carried out with SYBR-Green qPCR Super-Mix-UDG with ROX (Invitrogen) with the specific primers detailed in Table SVI in a CFX96 Touch™ Real Time PCR Detection System (BioRad), with one cycle of 95°C for 2 min and 40 cycles consisting in 95°C for 30 s and 60°C for 30 s. Expression values were normalized to UBQ10 and to the WT in deficiency conditions using the 2^−ΔΔCt method. Values represent the arithmetic mean ± standard deviation (SD) of three biological replicates (n = 3).