In vitro processing of pre-MIR167a

The full length pre-MIR167a sequence was PCR amplified from tomato leaf cDNA pool and cloned in a TA vector with NcoI and BstEII restriction sites on either end. Proper directionality of the clone was confirmed by sequencing. The construct was linearized by digesting with BstEII restriction enzyme, gel-purified, and 1μg of linearized DNA was transcribed using T7 RNA polymerase (NEB) in 20 µl reaction volume. The 238 bases transcript was used in subsequent processing experiments.

The total protein extract to be used in subsequent in vitro dicer activity assays was prepared from young leaves of 45 days old tomato plants kept under control (room temperature), cold or heat stresses. The plant leaves were crushed with liquid nitrogen and 2 ml of extraction buffer (20 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 5 mM DTT, and EDTA free protease inhibitor) cocktail was used for suspension of each gram of tissue. The cell debris was removed by centrifugation at 20,000 g at 4°C for 20 min.

In vitro transcribed pre-MIR167a transcript (4 µl) was incubated with the total protein extract (∼20 µg) in a 20μL reaction containing 4 μL of 5X dicer reaction buffer (50 mM NaCl, 6 mM MgCl2, 30 mM Tris – HCl of pH – 7.5, 5 mM ATP, 1 mM GTP), 20 U of Ribolock RNase inhibitor (Thermo) for 2 h at 26°C. The reaction was stopped by adding 200μL of TRIzol reagent, RNA was then purified and electrophoresed in a 8% Urea-PAGE containing 8M Urea, and transferred to nylon (Hybond XL) membrane. The membrane was hybridized with radiolabelled LNA probes against miR167a. Total RNA from a control tomato plant was also run in parallel lane to determine the position of mature miR167a. Radioactive signals were detected by using a phosphor imager as described earlier.