Cdna transcription kit

Total RNA was isolated using TRIzol reagent (Takara, Dalian, China). cDNA was obtained by reverse transcription using a cDNA transcription kit (Takara, Dalian, China). Real-time PCR was performed in 96-well optical plates on an ABI StepOne Plus Real-time PCR system using SYBR Premix Ex Taq™ (Takara, Dalian, China). The primers used for RT-PCR are shown in Table 2. Analysis of the relative gene expression level was achieved using the 2^-ΔΔCT method and gene expression levels were normalized to GAPDH.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA), and the concentration and purity of RNA were detected with the help of a UV spectrophotometer (Thermo Fisher Scientific, USA). cDNA was synthesized using a cDNA Transcription Kit (Takara, Tokyo, Japan). qRT-PCR was performed using the SYBR Green PCR Master Mix kit (Takara). The primer sequences of BDNF were as follows: forward, 5′-GGGA CCGGT TTG TGT-3′; reverse, 5′-TTG CTT TTT CAT GGG GGC A-3′. The primer sequences of BDNF–AS were forward, 5′-TACCACAAGGTACCAACCATATATG-3′, reverse: 5′-CATGTGGTTCTGTTTCAATGCCC-3′. GAPDH was the internal control, and the relative expression of BDNF-AS and BDNF was calculated by the 2^−ΔΔCt method.

Total RNA was isolated with TRIzol^® (Invitrogen) according to the manufacturer's protocol and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). A complementary DNA (cDNA) Transcription kit (TaKaRa, China) was applied to reverse transcribe total RNA into cDNA, then an SYBR Green Kit (TaKaRa, China) was used for qRT-PCR. Primer sequences were synthesized as follows: KLF6 (5′-ACGACCAAGTTTACCTCTGAC-3′ and 5′-CAGCCCCATAGTTGAGAAGAT-3′), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′-GGCTCTCTGCTCCTCCCTGTT-3′ and 5′-CTGTGCCGTTGAACTTGCCG-3′). qRT-PCR was performed on a Bio-Rad CFX96 system (Bio-Rad, USA). ΔCt was measured based on the difference in Ct values between the target gene and the housekeeping gene GAPDH, and the relative levels of the messenger RNA (mRNA) of target genes were calculated with the 2^−ΔΔCT method according to the manufacturer's instructions.