A total RNA extraction kit (Tiangen Biotech, Beijing, China) was used to extract total RNA from BV2 cells pretreated with VD11 for 12 hours. The RNA purity and concentration were measured using an ultra-microspectrophotometer (Thermo Fisher Scientific). The cDNA was reverse transcribed using a SureScript kit (GeneCopoeia, Guangzhou, China) in a total volume of 20 μL at 25°C for 5 minutes, 42°C for 15 minutes, and 85°C for 5 minutes, and then held at 4°C. A BlazeTaq kit was used for polymerase chain reaction (PCR) amplification (GeneCopoeia, Guangzhou, China). β-Actin (5′-TCA TCA CTA TTG GCA ACG AGC-3′, 5′-AAC AGT CCG CCT AGA AGC AC-3′), NGF (5′-TCT ATA CTG GCC GCA GTG AG-3′, 5′-GGA CAT TGC TAT CTG TGT ACG G-3′), and BDNF (5′-CTC CTG GGT TCC TGA GCA TC-3′, 5′-TTC ACT CCC TGA GTC ACA GC-3′) primers were purchased from GeneCopoeia. A Life Technologies thermocycler (Thermo, Waltham, MA, USA) was used to perform the qPCR reaction. The 2–ΔΔCt method was used to calculate the mRNA expression of NGF and BDNF, which was normalized to β-Actin (Yin et al., 2021).
Brain derived neurotrophic factor (bdnf)
Manufactured by GeneCopoeia
Sourced in China
BDNF is a protein that plays a critical role in the growth, development, and survival of neurons in the central and peripheral nervous systems. It is a member of the neurotrophin family of growth factors and is involved in various neurological processes, including synaptic plasticity, neurogenesis, and neuroprotection.
Automatically generated - may contain errors
2 protocols using brain derived neurotrophic factor (bdnf)
1
RNA Extraction and qPCR Analysis of Neurotrophic Factors
2
Profiling miRNA and mRNA Expressions
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The DNA/RNA isolation kit (Tiangen, Beijing, China) was used to extract total RNA. Primers were designed and synthesized by TIANGEN BIOTECH Co., LTD). The following primers were used: U6 forward, 5′-CTGGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACGAATTTGCGT-3′. has-miR-195-3p primer sequence: forward, 5′-CCAAUAUUGGCUGUGCUGCUCC-3′. RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Tiangen) following the manufacturer’s instructions. The following primes were used: BDNF (GeneCopoeia, Guangzhou, China) forward, 5′-GGCTTGACATCATTGGCTGAC-3′, reverse, 5′-GCCGAACTTTCTGGTCCTCAT-3′; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. The RT-qPCR reaction system (20 µL) contained 2 µL cDNA, according to the manufacturer’s instructions. U6 or GAPDH served as internal controls. The relative transcription levels of target genes were calculated using the 2−ΔΔCq method. ΔΔCq was calculated using the following equation: ΔΔCq = [Cq (target gene) − Cq (internal reference gene)] experimental group − [Cq (target gene) − Cq (internal reference gene)] control group.
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