Microrna rt kit

Collected tissues or cultured cells were isolated using miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). The concentration of obtained RNAs was detected with NanoDrop-1000 apparatus (Thermo Fisher). Then, cDNA was synthesized with a High-Capacity cDNA RT Kit (Thermo Fisher) or MicroRNA RT Kit (Thermo Fisher). For determining relative levels of RNAs/mRNAs, a SuperReal PreMix Color kit (Tiangen, Beijing, China) was employed with a Mx3000P system (Stratagene, Santa Clara, CA, USA). Obtained data were analyzed with the 2^-∆∆Ct method, and U6 and β-actin acted as references. The sense and antisense primers were circ-MBOAT2 5′-ATGCCTTACACTTTCTTG-3′ and 5′-GAGCTAGTTTTGCTTGAA-3′; linear MBOAT2 5′-GATGTTTCGGAAGGATGA-3′ and 5′-TTGTAAGAGCAAAGTGGG-3′; miR-433-3p 5′-ACACTCCAGCTGGGATCATGATGGGCTCCT-3′ and 5′-TGGTGTCGTGGAGTCG-3′; miR-144-3p 5′-ACACTCCAGCTGGGTACAGTATAGATGA-3′ and 5′-TGGTGTCGTGGAGTCG-3′; GOT1 5′-CTGGGAGTGGGAGCATAT-3′ and 5′-CAAGGGCAAGACGAGAAG-3′; U6 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 5′-GGTCACCAGGGCTGCTTT-3′ and 5′-GGAAGATGGTGATGGGATT-3′; β-actin 5′-CACCATTGGCAATGAGCGGTTC-3′ and 5′-AGGTCTTTGCGGATGTCCACGT-3′.

For extraction and purification of RNA, RNeasy Plus Mini Kit (Qiagen) was used according to manufacturer’s instructions, and for microRNA, miRNeasy Mini Kit (Qiagen) was used. Quantity was measured using NanoDrop. Reverse transcription of DNA-free RNA with random hexamer primers was performed using the “First strand cDNA Synthesis kit” according to manufacturer’s instructions (Fermentas) or MicroRNA RT kit (Life Technologies) using 10 ng RNA. Successful DNase I treatment of all RNA preparations was established by PCR analysis of the MYC promoter. qRT-PCR reactions were performed on the Step I qRT-PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix and assays for ACTG2 (Hs00242273_m1), GAPDH (Hs02758991_g1), hsa-miR-145 (002278) and RNU48 (001006) (Applied Biosystems). All samples were amplified in triplicates, and non-template controls were included. Each sample’s mean threshold value was corrected for the corresponding mean value for GAPDH mRNA or RNU48 miRNA, used as endogenous controls.

Total RNA was extracted using TriZol reagent (Life Technologies) and the miRNeasy kit (Qiagen, Hilden, Germany). Mature miRNA levels were quantified using MicroRNA RT kit and TaqMan miRNA assays (Life Technologies) for mir-155 and the housekeeping RNAs RNU44 and RNU48, following manufacturer's protocols. For amplification of mRNA molecules, TriZol extracted RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Life Technologies) and amplified using SensiFAST SYBR Green Master mix (Bioline, London, UK) on an ABI7900HT machine.

Total RNA was extracted using QIAzol reagent (Qiagen, Hilden, Germany) and the miRNeasy micro kit (Qiagen). Mature miRNA levels were quantified on an ABI7900HT machine using MicroRNA RT kit and TaqMan miRNA assays (Life Technologies) for miR-155-5p and the housekeeping RNAs RNU44 and RNU48, following manufacturer’s protocols.

miRNA validation was carried out using TaqMan real-time quantitative PCR (qPCR). Reverse transcription (RT) was performed using miRNA-specific stem-loop RT primers and a MicroRNA RT Kit (Life Technologies) following the manufacturer’s instructions. The resulting cDNA was diluted and used immediately for qPCR or stored at −20°C until use. miRNA expression levels were measured by real-time qPCR in a ViiA ⁷ Real-Time instrument (Life Technologies) using TaqMan^® Universal Master Mix (Life Technologies). The miRNA expression levels were normalized to those of cel-miR-39 and determined by the equation 2^–ΔCt, where ΔCt=cycle threshold (Ct) (miRNA) – Ct (cel-miR-39).