Midetect a track mirna qrt pcr starter kit

Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA). Reverse transcription and qPCR for miR-132-3p were performed using a miDETECTA Track™ miRNA qRT-PCR Starter kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions on a BioRad IQ5 system. Each value was normalized to that of RnU6. Reverse transcription and qPCR for the mRNA levels of FOXO1, GDF5 and SOX6 were carried out as described previously [49 (link)]. Expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and relative gene expression levels were calculated using the 2-ΔΔC_t method. All experiments were performed in triplicate. The primers were described in Table 1.

Reverse transcription of miRNAs and real-time quantitative reverse-transcription polymerase chain reaction- (qRT-PCR-) based quantification of miRNA levels were performed using the miDETECT A Track miRNA qRT-PCR Starter Kit (RiboBio, Tokyo, Japan). For PVT1 gene expression analysis, the first strand of cDNA was generated using a PrimeScript first-strand cDNA synthesis kit (TaKaRa, Tokyo, Japan). qRT-PCR was performed using the SYBR Premix Ex Taq II kit (Takara, Tokyo, Japan) on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA). The qPCR primers used in this study are listed in Supplementary Table 1.

We isolated total RNA including circulating IL-11 RNA using RNAiso Plus/Blood (Takara) per the manufacturer’s protocol and used 0.5 μg RNA to synthesize cDNA by PrimeScript RT reagent Kit (Perfect Real Time) (Takara). The concentrations of the RNA samples were determined by OD260 using a NanoDrop ND-1000 instrument. The resultant cDNA was used for PCR reactions using the following the manufacturer’s protocol. PCR annealing temperature varied depending on the primers used. To specifically detect the expression of target coding and circRNA genes, we used the SYBR Green method with primers described previously.^{64 (link)} GADPH was used as an internal control. Moreover, qRT-PCR for miRNAs was performed using miDETECT A Track miRNA qRT-PCR Starter Kit (Ribobio, China) following the manufacturer’s protocol. Delta-delta Ct values were used to determine their relative expression as fold changes, as previously described.^{65 (link)} The qRT-PCR results of expression levels of target genes shown as relative values.

The isolation of the total RNA from both cell lines and frozen tissues were used with TRIzol reagent (Ambion, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quantity and purity were evaluated by a Multiskan GO 1510 spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland). All quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) process was performed in a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). First, a miDETECT A Track miRNA qRT-PCR starter kit (Ribobio, Guangzhou, China) was used for synthesizing cDNA of small RNAs and evaluating miRNAs expression as previously reported.¹¹ (link)^,12 (link) Second, the synthesizing cDNA of mRNA was used with the ReverTra Ace qPCR RT master mix (Toyobo, Osaka, Japan) from 1 μg of total RNA, and the levels of mRNA expression were detected using PowerUp SYBR Green master mix (ABI/Life Technologies, Austin, TX, USA) according to the manufacturer’s protocol. The specific primers of miR-29s were synthesized by Ribobio (Guangzhou, China). The investigators were blinded to the results of clinical and pathological diagnoses. All targets and references gene were amplified in triplicate wells. The 2^−ΔΔCt method was used to calculate the relative levels of targets.

The total RNA was extracted from the NPCs using RNAiso (Takara, Kyoto, Japan). According to the manufacturer’s protocol, reverse-transcribed (RT) complementary DNA (cDNA) was synthesized with the Evo M-MLV RT Premix (Accurate Biology, Changsha, China). For qRT-PCR, a CFX96 Touch™ Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA) was used in conjunction with 2x SYBR Green qPCR Master Mix (Selleck, Houston, TX, USA). To quantify miR-302c expression, an RT reaction and qPCR were performed using a miDETECTA Track™ Uni-RT Primer and a miDETECTA Track™ miRNA qRT-PCR Starter kit (RiboBio, Guangzhou, China). GAPDH and U6 served as controls for mRNA and miRNA levels, respectively. The 2^−ΔΔCT method was used to calculate the relative expression levels. The primer sequences are listed in Table 2.

Total RNA was extracted from mouse eyes and cell lines using an RNAfast 200 total RNA rapid extraction kit (Feijie Biotechnology, Shanghai, China). After the quantity and purity were determined by NanoDrop 2000 (Thermo, Shanghai, China), 1 μg of total RNA was used to generate cDNA by miDETECT A Track miRNA qRT-PCR Starter Kit (RiboBio, Guangzhou, China). Quantitative RT-PCR analysis was performed using Blaze Taq™ SYBR Green qPCR Mix 2.0 (GeneCopoeia, Guangzhou, China) to detect the mRNA levels of interleukin (IL)-1β, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α. Primers used for qRT-PCR are listed in Table 1. The relative mRNA expression of each target gene was normalized to that of the housekeeping gene β-actin and GAPDH, and the result was fold-changed compared to the blank control group (set to 1).