RNA Extraction from Plant Tissues

The TRIzol-column hybrid method was used to extract RNA from needles, phloem, and root tissues following Untergasser’s protocol [22 ]. First, 500 µL of TRIzol was added to homogenized tissue and was incubated for 5 min at room temperature. Then, 100 µL of chloroform was added and mixed well, and the mixture was incubated for 2 min at room temperature. The mixture was then centrifuged at 12,000 rpm for 15 min at 4 °C. The aqueous upper phase was transferred to a new tube, and an equal volume of 70% ethanol was added and mixed well. The phase liquid was transferred to the RNeasy Mini spin column and was centrifuged at 12,000 rpm for 15 s, and the flow-through was discarded. Then, 350 µL of buffer RW1 was added to the column and centrifuged, and the flow-through was discarded. A total of 500 µL of buffer RPE was added to the column and centrifuged, and the flow-through was discarded. The buffer RPE step was repeated. The column was placed in a fresh collection tube and was dry centrifuged for 2 min at maximum speed. Finally, the column was placed in a new microcentrifuge tube and eluted with the appropriate amount (~50 µL) of RNase-free water.