Kapa qrt pcr kit

TRIzol purchased from Generay Biotech (Shanghai, China) were utilized to extract total RNA extraction from cells, and the concentration of extracts was measured by ultraviolet spectrophotometry. A PrimeScript® RT reagent Kit (Thermo Fisher, Massachusetts, USA) was used for the reverse transcription of the total RNA. 1 μL of the primers (10 μmol/L) of miR-429 synthesized and purified by Synbio Technology (Suzhou, China) were configured the reaction system according to the instruction of KAPA qRT-PCR kit (Sigma-Aldrich, Missouri, USA). The primer sequences of U6 and miR-429 are shown in Table 1.

The total RNAs were extracted from the tissues and cells with TRIzol reagent, and the concentrations of the extracts were measured by ultraviolet spectrophotometry. 1 μg of RNAs was reversed as cDNA with TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The primers were synthesized and purified by RiboBio (Guangzhou, China). 10 μL of reaction system was prepared according to the instructions of the KAPA qRT-PCR kit (Sigma-Aldrich, Missouri, USA). The reaction conditions included the denaturation at 95°C for 3 min, followed by amplification for 40 cycles at 95°C for 12 s, 56°C for 40 s, and 70°C for 30 s. The relative expression levels of miR-651-3p were calculated with the 2^−(ΔΔCt) method. The primers of miR-651-3p, ATG3, and U6 are listed in Table 1.

The levels of miR-124a and BRD4 in the whole blood and cells were measured by qRT-PCR. Total RNA was isolated from serum samples using a miRVana PARIS kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The cells treated with TRIzol reagent for extraction of total RNAs, and adherent cells were treated with trypsase before RNA extraction. After that, the total RNA in the extracts were transcribed as cDNAs by a PrimeScript® RT reagent Kit (Thermo Fisher, Massachusetts, USA). The primers of miR-124a and BRD4 synthesized by a PrimeScript® RT reagent Kit (Thermo Fisher, Massachusetts, USA) were used in the experiment. The reaction systems (10 μl) of qRT-PCR were prepared according to the operational instruction of a KAPA qRT-PCR kit (Sigma-Aldrich, Missouri, USA). U6 was used as the endogenous controls. The following conditions were used: denaturation at 95°C for 3 min, followed by amplification for 40 cycles at 95°C for 12 s and at 53°C for 40s, and 70°C for 30s. The relative levels of miRNA or mRNA were calculated with the 2^−(ΔΔCt) method [14 (link)]. The primer sequences of miR-124a, BRD4, and U6 are listed in Table 1.

Total RNAs of the tissues or cells were extracted by a TRIzol reagent. After that, the extracts were transcribed into cDNA by a PrimeScript® RT reagent Kit (Thermo Fisher, Massachusetts, USA). The primers of miR-1226-3p were synthesized and purified by Synbio Technology (Suzhou, China). According to the operation instruction of the KAPA qRT-PCR kit (Sigma-Aldrich, Missouri, USA), the reaction systems (10 μl) were prepared for qRT-PCR, and the reaction conditions included predenaturation at 95°C for 30 s, 95°C for 5 s, and 60°C for 30s for a total of 40 cycles. U6 was used as the control of miR-1226-3p. The sequences of forward and reverse primer of miR-612 and U6 have been shown in Table 1.