Anchored oligo dt 15

Total RNA was isolated from A. thaliana seedlings with trizol reagent (Ambion). RNA was quantified by UV spectrophotometry and its integrity was visually assessed on ethidium bromide-stained agarose gels. Total RNA (1.5 μg) was first converted into cDNA by reverse transcription (RT) using SuperScript II reverse transcriptase (Invitrogen) and anchored oligo(dT)₁₅ (Roche) and 18S reverse primer. PCR was performed under the following conditions to maintain a linear response in the range of the cDNA concentrations used (see Supplementary Table SIV for the primer-specific sequences): 30 cycles, except 20 cycles for 18S, of three temperature segments of 30 s (Td 94°C/Th 55°C/Te 72°C). The PCR products were visualized in 2% agarose gels. Real-time PCRs (qPCR) were carried out with SYBR-Green qPCR Super-Mix-UDG with ROX (Invitrogen) and specific oligonucleotides (Supplementary Table SV) in a StepOnePlus Real-Time PCR System (Applied Biosystems) under 1 cycle of 95°C for 2 min and 40 cycles consisting in 95°C for 30 s and 60°C for 30 s. The results correspond to the comparative Ct (cycle threshold) method (ΔΔCt). The UBQ10 gene was used as a loading control. Values are relative expression with respect to the first sample in each graph, in arbitrary units.