Primescript double strand cdna synthesis kit

Total RNA was extracted from tissues and cells using RNAiso reagents (Takara Bio, Inc.) according to the manufacturer's protocol. Total RNA was quantified using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, RNA (2 µg) was reverse transcribed into cDNA using the One Step PrimeScript miRNA cDNA synthesis kit (Takara Bio, Inc.) at 37°C for 20 min and 80°C for 2 min or the PrimeScript™ Double Strand cDNA Synthesis kit (Takara Bio, Inc.) at 65°C for 5 min and 42°C for 1 h. Next, qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme BioTech Co., Ltd.) with thermocycling conditions as follows: Initial denaturation at 95°C for 30 sec, 40 cycles at 95°C for 5 sec and 60°C for 20 sec. The sequences of the primers used for qPCR are presented in Table III. miRNA and mRNA expression levels were quantified using the 2^−ΔΔCq method (29 (link)) and normalized to the internal reference genes U6 and GAPDH, respectively.

N. benthamiana leaves after being infiltrated with agrobacteria containing the NbFKPPIase transient overexpression or silencing construct were collected for RNA extraction. A total amount of 1.5 µg RNA per sample was used for transcriptome analysis. Double‐stranded cDNA was synthesized using the PrimeScript Double‐Strand cDNA Synthesis Kit (Takara) following the manufacturer's recommendations. DNA was cleaned up with KAPA Pure Beads (KAPABiosystem; Roche) followed by end repair and A‐tailing reaction with the KAPA HyperPrep Kit (KAPABiosystems). DNA was cleaned up for adaptor ligation and subjected to sequencing with a Direct cDNA Sequencing Kit (Oxford Nanopore Technology). A total amount of 100 pmol of cDNA was loaded on Nanopore R9.4 flow cells and sequenced using the MinION platform (Oxford Nanopore Technology).

Total RNA was extracted from brain tissue by using TRIzol (Invitrogen) according to the manufacturer’s protocol. RNA was reverse transcribed by using a PrimeScript Double Strand cDNA Synthesis kit (Takara Bio Inc.), and first-strand products were used as PCR templates. Quantitative RT-PCR was performed by using a QuantiNava SYBR Green PCR kit and performed by using the iQ5 multicolor RT-PCR detection system (Bio-Rad Laboratories). The thermal cycling profile was as follows: 55°C for 30 min, 95°C for 15 min, and then 40 cycles of 95°C for 30 s, and 55°C for 30 s. The primer sequences used were as follows: BACE1 forward, 5′-TACTACTGCCCGTGTCCACC-3′; BACE1 reverse, 5′-ACAACCTGAGGGGAAAGTCC-3′; PS1 forward, 5′-CTCATGGCCCTGGTATTTATCAA-3′; PS1 reverse, 5′-GAGCCATGC GGTCCATTC-3′; ADAM10 forward, 5′-GTTGCCGCCTCCTAAACCA-3′; ADAM10 reverse, 5′-GGCGGTCTCCTCCTCTTTAAAG-3′; β-actin forward, 5′-AATGTGTCCGTCGTGGA TCT-3′; and β-actin reverse, 5′-GGTCCTCAGTGTAGCCCAAG-3′.