Primescript first strand cdna synthesis kit

m⁶A methyltransferase METTL3 expression in brain tissues of the NTD and control groups was detected by real-time qRT-PCR. Total RNA was extracted from brain tissues using TRIzol reagent (Invitrogen). Reverse transcription for cDNA synthesis was performed using the PrimeScript™ first strand cDNA Synthesis Kit (Takara, Beijing, China). Real-time qRT-PCR was conducted using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and the 7900HT Fast qPCR System (Applied Biosystems, Foster City, CA, USA). Cycling conditions were as follows: initial denaturation at 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 60 s, followed by a melt curve analysis from 60°C to 95.0°C in increments of 0.5°C per 10 s. The internal control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the relative expression levels of lncRNAs and genes were calculated using the 2^−ΔΔCT method.

The peach variety “Xiahui 5” is planted in the experimental base of Shandong Agricultural University, with fruit round, creamy yellow pericarp, more than 80% of the fruit surface with red, white meat, delicate flesh, ripe in mid-July, and a fruiting time of about 100 days [33 (link)]. Samples of leaves, stems, flowers, and fruits were collected. Total RNA was extracted from the samples using an RNA extraction kit (Tiangen, Beijing, China), and the RNA purity was determined using an ultra-micro UV analyzer to confirm that the OD260/OD230 ratio was between 1.8 and 2.1. First-strand cDNA was synthesized using the PrimeScript first-strand cDNA synthesis kit (Takara, Dalian, China). Real-time quantitative polymerase chain reaction (qRT-PCR) was performed on the ABI7500 system using the SYBR premix ExTaq (Takara, Dalian, China) with the following procedure: 95 °C for 5 min, followed by 45 cycles at 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 20 s. Reaction volume is 25 μL (including UItraSYBR Mixture (CWBIO, Taizhou, China) 12.5 μL, primer-F 0.5 μL, primer-R 0.5 μL, ddH₂O 10.5 μL, cDNA 1 μL), with three biological replicates per sample. The relative expression level was calculated by the 2^−ΔΔCT method [34 (link)]. Primers for qRT-PCR are listed in Table S1.

For qRT-PCR analysis, total RNA was extracted from 7-day-old seedlings using the RNeasy plant mini kit (Qiagen), and the first-strand cDNA was synthesized using the Takara PrimeScript First-strand cDNA synthesis kit (TAKARA). Next, PCR was performed on an Illumine Eco (Illumina) System with an SYBR probe (TAKARA). Expression levels of eIF4E1 were normalized to the expression level of two reference genes (ACTIN 2 and TUBULIN 2), respectively. The primers used are listed in Supplementary Table S1. Results obtained from three biological repeats with standard deviation (SD).

Total RNA was isolated from that obtained from the injured spinal cord (2-mm-long tissue samples) using an RNeasy Micro kit (Qiagen, Hilden, Germany). For cDNA synthesis, a reverse transcription reaction was performed using a Prime Script first-strand cDNA Synthesis kit (Takara Bio, Kusatsu, Japan). qRT-PCR was performed using primers that we’re interested in experiment and the SYBR Green Master Mix (Takara Bio, Kusatsu, Japan). The primer sequence is given in Table 1. The target mRNA level was normalized to the level of the GAPDH and compared with the control. Data were analyzed using the ΔΔCT method.

Total RNA from erastin-treated A549 and H1299 cells was extracted using TRIzol reagent (Life Technologies), and according to the manufacturer’s instructions, Primescript first-strand cDNA synthesis kit (Takara, Dalian, China) was used to turn the extracted RNA into cDNA. The SYBR-Green kit (Takara, Dalian, China) was applied to undertake real-time PCR in order to measure the levels of ferroptosis genes’ mRNA expression. The computation is based on the 2-∆∆CT approach, and β-actin was used as a comparison to normalize the expression and evaluate the relative expression of each group. The real-time PCR primers were purchased from Tsingke Biotechnology and are provided in Table S1.

Animals were killed 2 h post a single Flx (15 mg/kg) or saline injection by decapitation; the PFC was dissected out and snap-frozen in liquid nitrogen. RNA was extracted using Tri reagent (Sigma) and reverse transcribed using a cDNA synthesis kit (PrimeScript First Strand cDNA Synthesis Kit, Takara Bio). qPCR was performed with primers for the genes of interest (Supplementary Table S1) using a Bio-Rad CFX96 real-time PCR machine. Data were analyzed using the ΔΔC_t method, as described previously [17 (link)]. Normalization was done using hypoxanthine guanine phosphoribosyl transferase (Hprt), whose level was unaltered across experimental groups. Gene expression of IEGs analyzed included activity regulated cytoskeleton associated protein (Arc), brain-derived neurotrophic factor (Bdnf), early growth response gene 1-4 (Egr 1-4), FBJ osteosarcoma gene (Fos), FBJ murine osteosarcoma viral oncogene homolog B (Fosb), Fos-like antigen 2 (Fosl2), Homer scaffolding protein 1-3 (Homer 1-3), Jun proto-oncogene (Jun), Jun B proto-oncogene (Junb), Jun D proto-oncogene (Jund). qPCR analysis was also performed for the 5-HT_2A gene (Htr2a) to further confirm our genotyping results.