Genomic dna eraser

Randomly selected potential mRNAs and miRNAs were used to validate the sequencing data. The RNA samples used for cDNA synthesis were the same as those used for small RNA library construction. First-strand cDNA for targeted mRNA genes were synthesized using a PrimeScript RT reagent kit with a genomic DNA eraser (Takara, Tokyo, Japan), while Mix-XTM miRNA first-strand synthesis kit was used to generate miRNA cDNA (Takara, Tokyo, Japan). Later, using SYBR Advantage^® qPCR Premix (Takara, Tokyo, Japan), the assay was performed with quantitative real-time PCR (qRT-PCR) in a Lightcycler480 (Roche International Diagnostics Systems, Rotkreuz, Switzerland). MicroRNA specific primers were designed with primer premier5 software. The entire mature sequence (21–23 nts) of the miRNA was used as miRNA specific, 5′ primer while the 3′ primer for qPCR was the mRQ 3′ Primer supplied with the kit. Three biological replicates and two technical replicates were carried out for each mRNA. The beta-actin gene (F-CACTGGAATGGTCAAGGCTG; R-CTCCATGTCATCCCAGTTG) for mRNAs and U6 for miRNAs were used as internal control/reference using comparative Ct methods. The relative expression changes were calculated using the 2−∆∆Ct method. All primers are listed in the Tables S7 and S8.

Total RNA was extracted from liver tissue using Trizol reagent (Takara, Dalian, China), and quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher, USA). Complementary DNA was synthesized using the PrimeScript^TM RT reagent kit with a genomic DNA Eraser (Takara, Dalian, China) in accordance with the manufacturer's protocol. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed with SYBR Premix Ex Taq^TMII (Takara, Dalian, China) in the Bio-Rad CFX96 Thermocycler (Bio-Rad, USA). All the primers are listed in Supplementary Table 1.

Total RNA was isolated from cells using RNAiso plus (Takara Bio Inc., Shiga, Japan). The isolated RNA was reverse-transcribed into complementary DNA with PrimeScript RT reagent kit with a genomic DNA Eraser (RR047A, Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instruction. An aliquot of complementary DNA was amplified on a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) with KAPA SYBR fast qPCR master mix (KAPA Biosystems, Woburn, MA, USA). The reaction was run using the following program: 30 sec at 95°C, 40 repeats of 10 sec at 95°C, 10 sec at 63°C, and 15 sec at 72°C. The sequences of the specific primers (Integrated DNA Technologies, Inc., Coralville, IA) are listed in Table 1. The fold change in the relative mRNA level to β-actin was calculated based on the Ct (ΔΔCt) method [32 (link)].

Total RNA was isolated using TRIzol reagent from rice tissues, including the root, stem, leaf, sheath, anther, pistil, palea, lemma, and anther at different stages according to spikelet length and anther morphology^{63 (link)}. Approximately 1 μg of RNA per sample was treated by genomic DNA eraser (TaKaRa) and then used to synthesize cDNA with the PrimeScript RT reagent kit. Two microlitres of cDNA was used as a template for RT-PCR. All the primers for RT-PCR are listed in Table S1. Real-time quantitative PCR was performed with a LightCycler 480 (Roche). Amplification was conducted using the following protocol: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. ACTIN was used as an internal control. Each experiment was repeated three times, each with three replicates.

The gene expression change was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Then, total RNA of the mycelia was extracted by the plant RNA Kit (Omega Bio-tek, California, United States) and reverse transcribed using the PrimeScript^™ RT reagent kit with genomic DNA Eraser (TaKaRa, Tokyo, Japan) for complementary DNA. The expression of the selected genes was determined using SYBR Premix Ex Taq II (TaKaRa Tokyo, Japan) by RT-qPCR using the Stratagen Mx3000P (Agilent, California, United States). The β-actin gene was used as a reference gene. All primers used in this study were listed in Supplementary Table S1.