Transscript 2 one step gdna removal and cdna synthesis supermix

The WT plants and T₂ homozygous slpg mutants grown in a greenhouse were used to compare the expression levels of SlPG. Total RNAs were extracted from frozen tissue using TransZol by following the protocol provided by the product supplier (TransGen Biotech, Beijing, China). For reverse transcription, 2 μg of total RNA was used to synthesize first-strand cDNA using TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). For qRT-PCR, the reactions were performed using TransStart® Green qPCR SuperMix (TransGen Biotech, Beijing, China). The PCR amplification conditions begin with a denaturing step for 20 s at 95°C, followed by 40 cycles of 95°C for 5 s and a primer extension reaction at 60°C for 30 s. PCR products were monitored using Bio-Rad CFX connect (Bio-Rad). All PCR reactions were run with three biological replicates each. Data were analyzed using the 2^–ΔΔCt method. SlActin (solyc03g078400) was selected as the internal reference.

Total RNA was extracted with TRIzol (cat# 15596026, Invitrogen) and reversely transcribed using TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix (cat# AH311-03, TransGen Biotech). The qRT-PCR was completed using the SYBR Green Master Mix kit (cat# A25742, Thermo Fisher Scientific), and the assay was conducted with the QuantStudio5 Real-Time PCR System (Life Technologies). The primers are listed in Supplementary file 2.

RNA was extracted with Tripure™ reagent (Roche, Basel, Switzerland), and cDNA was synthesized with TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Mature miR-124a was quantified based on the stem-loop reverse transcript qPCR method using the artificially designed stem-loop-RT primer and qPCR primers. MiR-124a expression was normalized to snRNA U6 (RnU6). Meanwhile, cDNA transcribed through oligo (dT) was used for the analysis of other gene expression, and ACTB (β-actin) was used as an internal control. qPCR was performed using SYBR Green Mix (Roche, Indianapolis, USA) with a Roche LightCycler 480 II Real-Time PCR System. The primer sequences used here are listed Tables S1 and S2 in supplementary materials.

Two low-Pi-sensitivity extreme maize inbred lines P178 and 9782 were treated for 0, 4, 12, 24, 36, and 48 h with low-phosphorus stress at the seedling stage. In particular, samples were collected at the same time after different treatment times. The culture conditions of the materials were similar to those used in our previous studies [75 (link)]. Total RNA was extracted from plant materials using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using TransScript^®II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). The cDNA was diluted five-fold with nuclease-free water and used as a template for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. qRT-PCR was performed in three technical replicates using TransScript^®II Green One-Step qRT-PCR SuperMix (TransGen, Beijing, China). The expression of the housekeeping ZmUBI1 gene was used in maize and IPP2 in Arabidopsis was used as an internal control. The primers used are shown in Table S1.

Total RNA was isolated from ‘Tiny Padhye’ upper and basal tepals in four stages using an RNAprep Pure Plant Kit (polysaccharide & polyphenolic-rich) (TIANGEN, Beijing, China), according to the manufacturer’s instructions. Briefly, the first-strand cDNA was synthesized using TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) and oligo_(dT) primers. The qRT-PCR reactions were performed using Perfectstar™ Green qPCR SuperMix (TransGen Biotech, Beijing, China) and a Bio-Rad CFX96 system. The relative expression level of quantification was calculated based on the 2^−∆∆Ct formula method [49 (link)]. LilyActin was used as an internal control [50 (link)]. The primers for RT-qPCR were synthesized by Sangon Biotech (Shanghai, China; Table S1).

Total RNA was isolated using Tiangen RNAprep plant kit (Tiangen, Beijing, China) from underground shoots collected from two-year-old bamboo plants and treated with RNase-free DNase I (Tiangen). cDNA was converted from 1 μg RNA using TransScript^® II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech).
Luciferase reporter assays were conducted as reported [62 (link)] with slight modifications, including vector, restriction enzyme and plasmid. PeGRF6 sequence with the predicted target site was inserted into the pGreenII 0800-miRNA vector [63 (link)] between KpnI and EcoRI. The precursor sequence of ped-miR396d-5p was inserted behind the 35S promoter of the pCAMBIA1300 vector. pGreenII-0800::PeGRF6 and 35S::ped-miR396d-5p vectors were transformed into Agrobacteria GV3101 (pSoup) and GV3101, respectively. The transformed strains were co-infiltrated into the leaves of N. benthamiana. Empty pCAMBIA1300 vectors were used as the controls. At 48 h after infiltration, fluorescent signals were detected and compared. Table S5 shows the primers.

Meristems from approximately four-week-old plants were dissected within 2 hours of collection and immediately fixed in 100% acetone, followed by vacuum infiltration (0.06 MPa for 40 min)^{38 (link)}. Approximately 60 sympodial inflorescence meristem and floral meristem (SIM and FM) tissues were microdissected under a stereomicroscope using tweezers, dried for 3 min at room temperature to remove the remaining acetone, and immediately frozen in liquid nitrogen. The materials were thoroughly ground in an MM300 mixer mill (Retsch) with a tungsten bead (3-mm diameter). Total RNA was extracted from each sample using a PicoPure RNA Extraction kit (Thermo-Fisher, 12204-01). Gene expression analysis was performed as the previously reported method with some modifications^{39 (link)}. In brief, reverse transcription was performed with TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AH311-02) using 100 ng of total RNA. Quantitative PCR (qPCR) was performed with Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712-02) on a Bio-Rad CFX-96 Real-Time PCR instrument using the following program: 3 min at 95 °C followed by 40 cycles of 20 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C. UBI3 (Solyc01g056840) was used as the reference transcript for RT-qPCR. The primers used for RT-qPCR are listed in Supplementary Data 3.