Gotaq master mix

To confirm DNA amplifiability, the 16S rRNA gene was amplified using primers Eub338 and Eub518 (Fierer et al., 2005 (link)). To check for the presence of symbiotic marker genes (nifH and nodC) in the isolated nodules DNA, primers for nifH (Poly et al., 2001 (link)) and nodC genes (Sarita et al., 2005 (link)) were used for PCR amplification. The same PCR conditions were used for all three amplicons (16S, nifH, and nodC) which is as follows: 1 cycle of 95°C for 5 min (preheating), 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and a final extension period at 72°C for 5 min. The size of the PCR product was confirmed by electrophoresis on a 2% agarose gel. For PCR reactions, nuclease-free PCR grade water, 2x GoTaq Master mix, and polymerase were purchased from Promega.

Total RNA was extracted from rice tissues using an RNA Extraction kit (MG Med, Seoul, Korea), according to the manufacturer’s instructions. For synthesis of first-strand cDNA, 2 μg of total RNA was used for reverse transcription (RT) in 20 μL volume with oligo(dT)₁₅ primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA). For quantification of miR164b, stem-loop pulsed RT was conducted from 2 μg of total RNA in 20 μL volume using a miR164b-specific stem-loop primer and M-MLV reverse transcriptase (Promega) with the following conditions: 16 °C for 30 min followed by pulsed RT of 40 cycles at 16 °C for 2 min, 42 °C for 1 min, and 50 °C for 1 s, and then inactivation of reverse transcription at 70 °C for 5 min [57 (link)]. All RT products were diluted with 80 µL distilled water.
qPCR was performed with gene-specific primers and normalized to UBIQUITIN5 (UBQ5) (Os01g22490) or rice U6 snRNA (Table S1) according to the 2^−ΔΔCt method [58 (link)]. The 20 µL reaction mixture included 2 µL cDNA from RT or stem-loop pulsed RT, 1 µL 0.5 µM primer, and 10 μL 2X GoTaq master mix (Promega). qPCR amplifications were conducted with a LightCycler 480 (Roche, Basel, Switzerland) using the following program: 94 °C for 2 min followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min.

Total RNA was isolated using the RNeasy Plus Mini Kit, and 500 ng RNA per sample was reverse transcribed to cDNA in 25 μl reaction using the High-Capacity cDNA Reverse Transcription Kit. The RT reaction was performed at 25 °C for 10 min, followed by heating at 48 °C for 30 min, and then 95 °C for 5 min. The primers for human PEPD and GAPDH were purchased from IDT. The PEPD primers are as follows: 5′-CTGCAGGGCGGGGAGGAGAC-3′ (forward), and 5′-CGCCCCGGGAGTAGCAGTAGTG-3′ (reverse). The GAPDH primers are as follows: 5′-CCAGGGCTGCTTTTAACTC-3′ (forward), 5′-GCTCCCCCCTGCAAATGA-3′ (reverse). Each PCR amplification was carried out in 20 μl volume, containing 10 μl 2x GoTaq Master Mix (Promega, Cat# M7122), 0.5–1 μl of the reverse-transcribed mixture (cDNA), and 0.25 μM each of specific forward and reverse primers. The PCR condition for PEPD is as follows: 94 °C for 3 min, 35 cycles at 95 °C (denaturation) for 30 s, 61 °C for 30 s (annealing), and 72 °C for 35 s (extension), with the final extension performed at 72 °C for 5 min. The PCR condition for GAPDH is as follows: 94 °C for 3 min, 25 cycles at 94 °C (denaturation) for 30 s, 60 °C for 30 s (annealing), and 72 °C for 30 s (extension), with the final extension performed at 72 °C for 5 min. The PCR products were analyzed by electrophoresis with 1% agarose gel, stained by ethidium bromide, and visualized under UV light.