Realuniversal color premix sybr green kit

Total RNA was extracted from the uteri and endometrial cells using TRIzol (Invitrogen) according to manufacturer's instructions. The quantity of RNA was examined using a NanoDrop2000 instrument (Thermo Fisher Scienti c, Waltham, MA, USA). For mRNA detection, total RNA (2 μg) cDNA was synthesized using a FastKing gDNA Dispelling RT SuperMix kit (TIANGEN BIOTECH, Beijing, China). Gene expression was assessed by qPCR with 2μl of the synthetized cDNA using a Real Universal Color PreMix (SYBR Green) kit (TIANGEN BIOTECH). For miRNA detection, total RNA (1 μg) was used to synthesize cDNA using a miRcute Plus miRNA First-Strand cDNA kit (TIANGEN BIOTECH) and miR-192-5p was quali ed by qPCR using miRcute Plus miRNA qPCR (SYBR Green) kit (TIANGEN BIOTECH). The qPCR reactions were performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). GAPDH/U6 were set as the normalizing control. Relative quantities were calculated using the 2 -△△CT method. The sequences of all primers used are listed in Table 1.

The DEcircRNAs and DEGs targeted by the DEmiRNAs were retrieved according to starBase 16 . Moreover, the prediction results of TargetScan, miRTarBase and miRDB were integrated by starBase [17] (link)[18] [19] (link) . The candidates searched by three databases were intersected with the most important DEmiRNAs. Finally, the DEcircRNA-DEmiRNA-DEGs ceRNAs network was constructed and visualized using the R software.
Quantitative real-time PCR (qRT-PCR) of DEmiRNAs and TIMP1 in CRC and normal tissue Total RNAs was prepared from colonic tissue using an RNA extraction kit (TIANGEN BIOTECH, Beijing, China) according to the manufacturer's instruction. The extracted RNA was were performed to synthesize cDNA by using FastKing one-step kit (TIANGEN BIOTECH, Beijing, China). qRT-PCR was performed using RealUniversal Color PreMix (SYBR Green) kit (TIANGEN BIOTECH, Beijing, China) to assess the expression of target genes. U6 was used as an internal control for DEmiRNAs. GAPDH was used as internal control for TIMP1. In addition, the relative expression of RNAs was quanti ed by using the 2 -ΔΔCq method.