Total rna extraction kit

For the confirmation of pcdhg overexpression (Figure 3A and B), the brains of mice electroporated at P11–14 were promptly removed at sacrifice and placed into 4°C choline-aCSF. The specific target region was then identified using a fluorescent microscope, and approximately 500-μm-thick coronal slices were prepared. Tissue blocks containing the fluorescently labeled region, as well as the contralateral control side, were extracted from the same coronal slice and immediately transferred into an RNA extraction buffer.
For Pcdha knockout confirmation (Figure 2—figure supplement 2D), the semi-cortex of P11 mice was isolated and transferred into an RNA extraction buffer. Tissue RNA was extracted using the Total RNA extraction Kit (R4011-02, Magen) and then reverse transcribed using the PrimeScript RT Master Mix (RR036A, Takara).
Primers, the sequences of which can be found in the primers list (Supplementary file 1), were designed with the assistance of PrimerBlast (NCBI). SYBR Green Real-time PCR Master Mix (QPK-201, Toyobo) and the LightCycler 480 II (Roche) were employed for qPCR.
The expression level was calculated as $2^{Ct\left(Pcdhg\right)-Ct\left(GAPDH\right)}$ , and the normalized expression level (Figure 3B) was determined by dividing the expression level of the target fluorescent region by the expression level of the contralateral control region.

According to gene sequence of CAV-1 from rats in Gene Bank (156–692,536 bp), the primers of CAV-1 for polymerase chain reaction (PCR) amplification were synthesized in Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Total RNA was extracted from rat brain through Total RNA Extraction Kit (Magen, Guangzhou, China) according to the manufacturer's instructions. The reverse transcription reaction of the extracted RNA was conducted by using reverse transcription kit (GeneCopoeia, Inc., Rockville, MD, USA) to obtain cDNA according to the manufacturer's protocols. Totally 30 μL of DNA was purified by using Universal DNA Purification Extraction Kit (TIANGEN Biotech Inc., Beijing, China) according to the manufacturer's instructions. The pEGFP-C1 vector and purified CAV-1 gene product were subjected to double digestion by restriction endonucleases EcoR I and Sal I. The digested products were purified and harvested by electrophoresis. The CAV-1 gene was spliced into linear pEGFP-C1 vector to obtain pEGFP-C1-CAV1. The recombinant pEGFP-C1-CAV1 plasmid was subjected to extraction, double digestion by EcoR I and Sal I, identification, and sequence analysis. The validated pEGFP-C1-CAV1 plasmid was transferred into hippocampal neuronal cells for coming experiments.

Total RNA was separately isolated from the strawberry tissue of fruits in the Total RNA Extraction Kit (Magen) by the manufacturer's protocols. The purity and integrity of the RNA were determined by gel electrophoresis and the ratios of A260/A230 and A260/A280. To generate first-strand cDNA, 400 ng of total RNA was reverse transcribed using the HifairⅡ 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen) by the manufacturer's protocols.