Primescript 2 1st strand cdna synthesis kit

RNAs were extracted from immature maize seeds collected 6, 9, 14, and 20 DAP. Genomic DNA was digested using RNase-free DNaseⅠ(Fermentas, Ontario, Canada). cDNA synthesis and quantitative real-time PCR (qRT-PCR) of miRNAs were performed using the All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, Maryland, USA). U6 was used as an internal control. For qRT-PCR of coding genes, PrimeScript^TM Ⅱ1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) and SYBR Green FS Universal SYBR Green Master (Roche Applied Science, Indiana, USA) were used. PCR was carried out on the CFX96^TM Real-Time PCR Detection System (Bio-Rad, California, USA). The thermal cycling program consists of an initial denaturation step at 95°C for 10 min, then 40 cycles at 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s. Target gene abundance in each sample was normalized according to the U6 expression (for miRNAs) or TUBULIN expression (for coding genes) levels using the formula ΔCt = Ct (target gene)–Ct (U6) or Ct (TUBULIN). The experiment was performed with three biological repeats, each with three technical repeats. The primers used for qRT-PCR are listed in S4 Table.

Total RNA was isolated from SH-SY5Y cells using RNAiso Plus (TaKaRa, Gunma, Japan). Total RNA (2 μg) was reverse transcribed to cDNA using the PrimeScript^TM Ⅱ 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) to determine the mRNA expressions of α-syn through qRT-PCR using the SYBR Green reagent (TaKaRa, Japan). The PCR condition was as follows: 95 °C for 5 min, 60 °C for 20 s, and 40 amplification cycles. Housekeeping gene b-actin served as an internal control. Data analysis is based on the ΔΔCt method with the normalization of raw data to β-actin. Each reaction was run in triplicate. a-Syn primer: forward, forward: 5′-AGGAATTCATTAGCCATGGATGTATTC -3′; reverse: 5′-AGATATTTCTTAGGCTTCAGGTTCGTAGT-3′; β-actin primer: forward: 5′-GACCCAGATCATGTTTGAGA-3′; reverse: 5′-GCTTGCTGATCCACATCTGC-3′.

Total RNA was extracted individually from root, stem, leaf, developing embryo, and endosperm as well as young tassel and young ear. Reverse transcription was conducted using PrimeScriptTM Ⅱ 1st strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) with Oligo-T as primer. The full-length CDS was amplified by the TransStart FastPfu Fly DNA Polymerase kit (TransGen) using 1st strand cDNA as a template. Quantitative real-time PCR analyses were conducted through a 7500 Fast Real-Time PCR System (Applied Biosystem, Foster city, CA, USA) using SYBR green detection protocol (TaKaRa). Gene-specific primers and ubiquitin gene primers for the control standard were used in the RT-qPCR. The primer sequences used in cDNA synthesis and RT-qPCR are listed in Table S2.

Transgenic mice were genotyped with genomic DNA extracted from tail tips using the Viagen DirectPCR DNA Extraction System (Viagen). The genotyping primers are provided in Supplementary Table 2.
Total RNA was extracted from the cochlea with the anlage of the stria vascularis, the modiolus, and the tectorial membrane removed using TRIzol (Ambion). cDNA synthesis and qPCRs were performed with the PrimeScript^TM II 1st Strand cDNA Synthesis Kit (Takara) and TB Green^TM Premix Ex Taq^TM II (Takara). Each PCR was carried out in triplicate, and the relative quantification of gene expression was performed using the ΔΔCT method with Gapdh as the endogenous reference. Primer pairs were designed using the online Primer3 software, and sequences are provided in Supplementary Table 3.

Primer Premier 5.0 software was used to design primers, and the primers used in confirming T-DNA insertion sites are listed in Supplementary Table S1. Total RNA was extracted from tissues using a RNAprep Pure Plant Kit (Tiangen Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using a PrimeScript^TM II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian). Synthesized cDNAs were used for RT-qPCR with a SYBR Premix Ex Taq kit (TaKaRa, Dalian, China) on an ABI 7500 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, United States). Determination of relative changes in gene expression levels was based on three biological replicates. Primers were designed using the GenScript real-time PCR (TaqMan) primer design tool¹ (Primers are listed in Supplementary Table S1). The rice UBQ5 gene (LOC_Os03g13170) was used to normalize cDNA quantity.

Total RNA was extracted from the liver tissue of triploid rainbow trout by using a total RNA extraction kit (DP419, Tiangen, China). A cDNA template was obtained using the PrimeScriptTM II1st strand cDNA Synthesis Kit (6210A, TaKaRa, Japan).