Bulgeloop mirna qrt pcr primers

Total RNA was isolated from tissues or cells using Trizol (Invitrogen, USA) according to manufacturer’s recommendations. Then, RNA (1 μg) samples were reverse transcribed into complementary DNA (cDNA) using the Prime Script Reverse Transcription Kit (Takara Bio, Inc., Otsu, Japan). We then designed and purchased Bulge-loop™ miRNA qRT-PCR primers (one RT primer and a pair of qRT-PCR primers for each set) specific for miR-328-5p from RiboBio (Guangzhou, China). Quantitative polymerase chain reaction (qPCR) was performed using gene-specific primer sets and SYBR Premix Ex Taq (Takara Bio, Inc.) in an ABI StepOne Plus system (Applied Biosystems, CA, USA). The reaction conditions were 1 cycle of 95°C for 30 s followed by 45 cycles of 95°C for 5 s and 60°C for 30 s. All samples were analyzed in triplicate. Each experiment was repeated three times. The expression levels of target genes relative to β-actin were determined relative to β-actin by the 2^−ΔΔCt method. The expression levels of miRNAs were normalized to U6. The qPCR primer sequences used in this study are shown in Supplementary Table 1.

Total RNA was extracted with TRIzol reagent (Invitrogen, USA) and isopropanol precipitation methods. The extracted RNA was quantified by measuring the absorbance at 260 nm with a PuXin UV/visible spectrophotometer TU-1810. For miRNA level detection, the hsa-miR-664a-3p RT-qPCR was carried out using a Bulge-Loop miRNA qRT-PCR Starter Kit from Ribo Biotechnology (Guangzhou, China) with the provided miRNA reference gene (u6). Hsa-miR-664a-3p and u6 RT-specific forward and reverse primers and specific bulge-loop miRNA qRT-PCR primers were designed and purchased from Ribo Biotechnology (Guangzhou, China). For mRNA detection, total RNA (1μg) was removed DNA and performed Reverse transcription with Hifair Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR. All real-time PCR analyses were performed using gene-specific primers and the LightCycler® 480 real-time PCR system (Roche) using SYBR Premix Ex TaqTM (TaKaRa, RR420A). The relative amounts of each mRNA were analyzed using the comparative CT method, as described by Schmittgen and Livak49. All data are expressed as the mean ± SE of n = 3 independent repeats. All statistical analyses were performed using a two-tailed, paired Student's t-test, and P< 0.05 was considered significant. Primer sequences were listed in Supplementary Table 1.

Expressions of miRNAs were analyzed by qPCR using bulge-loop miRNA qRT-PCR primers (Ribobio, Guangzhou, China). U6 was chosen as an internal control to correct for analytical variations. Five differentially expressed genes from RNA seq data CPEB3, FGFR3, SUCLA2, ABHD3 and MUSTN1, were selected for qPCR and analyzed and normalized with the reference gene β-actin. Real-time qPCR primers were designed using the Premier Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The concentration of each primer was 20 μM. qPCR was performed on the Bio-Rad S1000 (Bio-Rad, Hercules, CA, USA) with SsoFast Eva Green Supermix (Bio-Rad) as follows: 95°C for 2 min; and 95°C for 10 s, 56°C for 30 s; and 72°C for 30 s for 40 cycles. Each reaction was performed in triplicate, and the data were analyzed by the 2^-△△Ct method.

Total RNA was isolated from breast cancer cells with RNAiso Plus (TaKaRa, Kusatsu, Japan). The extracted RNA was synthesized into cDNA with a PrimeScriptTM RT reagent Kit (#RR037A, TaKaRa) to produce a template suitable for qRT-PCR. For mRNA reverse-transcriptase PCR (RT-PCR), random hexamer primers were used, and for miRNA RT-PCR, specific primers (Ribobio) were used.
qRT-PCR was used to detect the expression of MICA, MICB, pri-miR-17-92 and c-Myc, with hypoxanthine phosphoribosyltransferase 1 (HPRT1) as an internal normalized reference. The sequences of the PCR primers are listed in Supplementary Table 4 Mature miRNA expression was quantified by qRT-PCR with specific BulgeLoop miRNA qRT-PCR primers (Ribobio), with U6 small nuclear RNA as an internal normalized reference (Supplementary Table 5). qRT-PCR was performed in a LightCycler 480II system (Roche Diagnostics, Basel, Switzerland) with a SYBR Premix EX Tag kit (#RR420A, TaKaRa). Relative expression was calculated as 2^-ΔCt or 2^-ΔΔCt after normalization with the reference control.

Total RNA was extracted from each sample using TRIzol reagent (Thermo, Waltham, Massachusetts, USA), separated using chloroform and precipitated with isopropanol according to the manufacturer’s instructions. Then, reverse transcription was performed using the PrimeScript RT Master Mix Kit (Takara, Tokyo, Japan) to acquire cDNAs. RT–qPCR was performed in a 20 µl reaction system using TB Green Premix Ex Taq (Takara, Tokyo, Japan) according to the manufacturer’s instructions. miR-210-3p and U6 were reverse-transcribed using Bulge-Loop miRNA qRT–PCR Primers (Ribobio, Guangzhou, China). GAPDH and U6 were used as internal references for mRNA and miRNA quantification, respectively. The primer sequences are listed in Table S2. Relative gene expression was quantified using the 2^−ΔΔCT method.