Lightcycler 480ii real time pcr system

Total RNA was extracted from the neuroepithelial cysts with TRIzol reagent (Invitrogen) and chloroform, precipitated with isopropanol, washed by 70% RNase-free ethanol and reverse transcribed into cDNA. 1 μg RNA were used to reverse to cDNA with the ReverTra Ace^® qPCR RT Mater Mix (Code No.FSQ-301) according to the manufacture’s protocol. Then 10 ng cDNAs products were used for qRT-PCR experiments with SYBR^® Green Realtime PCR Master Mix (Code No.QPK-201) and performed on the LightCycler^® 480 Ⅱ Real-Time PCR System (Roche). Relative expression of each target was calculated with human ACTB (which encodes beta-Actin) as a benchmark based on the delta Ct method. All qRT-PCR experiments were carried out in at least three replicates. Comparison between samples was performed using a Student’s t test. A p-value < .05 was considered statistically significant. The primers used in the qRT-PCR assays are listed in Supplementary Table S2.

RNA was isolated using the EASYspin Plus Plant Quick RNA isolation Kit (RN38) (Aidlab Biotechnology, Beijing, China) according to the manufacturer’s instructions. The first strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). The primers of RT-qPCR were designed by BatchPrimer3v1.0 (http://batchprimer3.bioinformatics.ucdavis.edu/index.html) and the Actin (1) reference gene was used as the internal control [49 (link)]. The primers used for RT-qPCR are listed in Table S1. RT-qPCR was carried out using a LightCycler 480Ⅱ real-time PCR system (Roche, Switzerland) using the RealUniversal Color PreMix (SYBR Green) (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Three biological replicates were performed for each sample. The relative expression levels were calculated using the comparative 2^−△△CT method [50 (link)].

The total miRNA from EVs or IEC6 cells was extracted using miRcute miRNA Isolation Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions and then the total RNA was reverse transcribed using a miRcute Plus miRNA First-Strand cDNA Kit (Tiangen, Beijing, China). The mature miRNAs were quantified by quantitative real-time PCR using the miRcute Plus miRNA qPCR Kit (SYBR Green). Briefly, quantitative real-time PCR was performed on a LightCycler 480Ⅱ Real-Time PCR System (Roche, Penzberg, Germany) containing 2 × miRcute Plus miRNA PreMix (5 μL), specific primer (0.2 μM for each primer), reverse-transcribed cDNA (1 μL), and RNase and DNase-free water ddH₂O (3.6 μL). Amplification was performed as follows: 95 °C for 15 min, 40 cycles at 94 °C for 20 s, 60 °C for 34 s; and a melting curve analysis was performed from 65 °C to 95 °C to confirm specific PCR products. The 5′-specific primers used for detecting miRNAs are listed in Table S1. Finally, the relative expression of miRNA was normalized against U6 levels and the expression levels were analyzed using the 2^−∆∆CT method [87 (link)].