Prime a gene labeling kit

Total RNA was isolated from 8-week-old flower buds using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. RNA was treated with DNase Max (QIAGEN) when RNAs were used in reverse transcription (RT)-PCR or quantitative RT-PCR (qRT-PCR) assays. Mitochondrial mRNA abundances were measured by qRT-PCR. They were calculated using the comparative ΔΔCt method after normalization to the nuclear 18S ribosomal RNA as previously described in (15 (link)). Two biological and three technical repeats were performed for these analyses.
For RNA gel blotting, 10 μg of total RNA was electrophoretically separated in formaldehyde-containing (1.5% [w/v]) agarose gels and transferred onto nylon membranes (Genescreen) as described previously (10 (link)). Hybridization probes were generated by PCR amplification using gene-specific primers (listed in Supplementary Table S1) and radiolabeled using the Prime A Gene labeling kit (Promega) according to the manufacturer's recommendations.

Total RNA was extracted from flower buds using the TRIzol reagent (Thermo Fisher Scientific), following the manufacturer’s instructions. To remove genomic DNA contamination from total RNA preparations, the DNase Max Kit (Qiagen) was used. For quantitative RT-PCR analysis, either 35 ng of total RNA was reverse-transcribed and amplified in a single tube with a volume of 15 µl using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) or reverse transcription was performed separately on 1 μg of the total RNAs using a random hexamer. Quantitative RT-PCR for analyzing splicing efficiency in plant mitochondria was carried out, as previously described in Haili et al.^{54 (link)}, using the described set of primers. Two biological repeats and three technical repeats were performed on each genotype. cRT-PCR was performed as described in Forner et al.^{24 (link)}. RNA gel blot analysis was performed as previously indicated in Haili et al.^{54 (link)}. Shortly, 10 µg of total RNA was separated on a 1.5% (w/v) agarose gel. After electrophoresis, the RNAs were transferred to nylon membranes and cross-linked in a UV Stratalinker. Hybridization was performed with specific DNA probes amplified by polymerase chain reaction (PCR) and radiolabeled by the Prime A Gene labeling kit (Promega), following the manufacturer’s instructions.

For cleavage fragment analysis, 20 μg of total RNA was loaded onto a 1% denaturing agarose gel and separated for 3–5 h at 120V. The RNA was blotted to an Amersham Hybond-NX nylon membrane (GE Healthcare Life Sciences) and UV-crosslinked (254 nm). After pre-hybridization in PerfectHyb^TM Plus Hybridization buffer (Sigma) for 1 h at 65ºC, the radioactively labeled probe (Prime-a-gene labeling kit, Promega) was hybridized overnight at 65ºC. Sequences for primers used for amplification of probe templates for GFP and endogenous miRNA targets are listed in Supplementary Table S4. After hybridization, the membrane was washed 3 times in 2xSSC (0.3 M NaCl, 30 mM sodium citrate), 0.1% SDS at 65ºC and developed by phosphorimaging. For Terminator (Epicentre) reactions, 10 μg of total RNA was incubated with 1 U Terminator (Epicentre) for 1 h at 30ºC. The reaction was terminated by phenol extraction and subsequent ethanol precipitation. The RNA was resuspended in 50% formamide and loaded with untreated RNA as control on a 1% denaturating agarose gel. Poly(A) RNA was purified from 10 μg of total RNA using Dynabeads Oligo(dT)₂₅ (Life Technologies) according to the manufacturer's instructions.