Mircute enhanced mirna qpcr kit

Total RNA in cultured BV2 cells was isolated in TRIzol (Beyotime, Shanghai, China). 200 ng total RNA was used as the template for reverse transcription using the Fastking RT kit (KR116; TIANGEN, Beijing, China) (for mRNA) and miRcute miRNA cDNA first strand synthesis kit (KR201; TIANGEN) (for miRNA). Quantitative PCR was performed with the Quant One-Step qRT-PCR kit (FP303; TIANGEN) and miRcute enhanced miRNA qPCR kit (FP411; TIANGEN) on an ABI 7900 real-time PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH mRNA and U6 small nuclear RNA (U6) were used as the internal control to NeuroD1 mRNA and mature miR-137-3p, separately. The reactions were performed in triplicate for each sample in at least three independent runs, and the primers involved were as follows: mmu-miR-137-3p, 5′-TGACAGCGGTAGCAGAGGCAGAG-3′ (sense) and 5′-CCGCTG CCCGCCTGCCGCTGGTA-3′ (anti-sense); NeuroD1, 5′-CGAAUUUGGUGUGGCUGUA-3′ (sense) and 5′-UACAGCCACACCAAAUUCG-3′ (anti-sense); U6, 5′-CGCTTCGGCAGCACATATAC-3′ (sense) and 5′-TTCACGAATTTGCGTGTCAT-3′ (anti-sense). GAPDH, 5′-AGAACATCATCCCAGCGT-3′ (sense) and 5′-AGCCTTCACTACCCTCTTG-3′ (anti-sense). The expression levels of NeuroD1 mRNA and miR-137-3p were analyzed using the threshold cycle 2^−ΔΔC_t method. Results are presented as fold change normalized to the control group.

Total RNA was extracted from tissues or cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and the concentration and purity of total RNA were determined using the Nanodrop 2000 spectrophotometer (Invitrogen; Thermo Fisher Scientific). Total RNA was reverse-transcribed into cDNA using the FastKing RT Kit (TIANGEN, Beijing, China). Then, the expression levels of CCDC144NL-AS1 and HMGA2 were determined via quantitative PCR using the Quant One-Step qRT-PCR Kit (TIANGEN). GAPDH served as the internal reference for CCDC144NL-AS1 and HMGA2 expression. To quantify miR-490-3p expression, cDNA was synthesized using the miRcute miRNA cDNA First-Strand Synthesis Kit (TIANGEN) and then subjected to quantitative PCR using the miRcute Enhanced miRNA qPCR Kit (TIANGEN). The expression level of miR-490-3p was normalized to that of U6 small nuclear RNA. All data were analyzed using the threshold cycle 2^−ΔΔCt method.