Micrornas qpcr kit

A total of 34 whole blood samples (including 16 RA without drug treatments and 18 control samples with healthy state) were collected from Fuzhou Second Hospital affiliated to Xiamen University. The Ethical Committee of Fuzhou Second Hospital affiliated to Xiamen University approved this study, and the respective patient provided informed consent in a written form. All whole blood samples were immediately frozen in liquid nitrogen after the collecting process and stored at −80°C. The extraction of total RNA was performed with the use of Trizol reagent (TAKARA, Dalian, China). With a miRNA First Strand cDNA Synthesis Kit (Sangon, China), the reverse transcription of total RNA and miRNAs was performed. Besides, this study adopted the MicroRNAs qPCR Kit (Sangon, China) for examining miRNA and mRNA expressions, with the following primers: GAPDH (forward: 5′-GACAGTCAGCCGCATCTTCT-3′, reverse: 5′-ACCAAATCCGTTGACTCCGA-3′), LSP1 (forward: 5′-CTGTTAGCTTGGGAAGAGG-3′, reverse: 5′-ATAGCCCCTCTCAGATAGTC-3′), MEOX2 (forward: 5′-ATACTAGGGGAGATTCTCGC-3′, reverse: 5′-TAGGACTTTGGAGGGCTTAG-3′), and GNLY (forward: 5′-TCTGGTCCTAACTCTACTGG-3′, reverse: 5′-CAATCCTAGACAGTGTAGGC-3′) synthesized by Sangon Biotech. GAPDH was then handled as an internal reference. The relative expression was calculated using the 2−ΔΔCT method. P values < 0.05 showed statistical significance.

Under the protocol of the manufacturer, TRIzol reagent ((Thermo Fisher Scientific, Inc.) was used to extract RNA from cervical cells and tissues. The concentration and quality of RNA were detected using NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Then mRNA and miRNA were reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara, Dalian, China). Furthermore, qRT‐ PCR analysis was performed to detect ACTA2‐AS1 and SMAD3 expression using SYBR Green PCR Master Mix according to the manufacturer’s protocol (Applied Biosystem, Waltham, MA). The miR-143-3p expression was tested by microRNAs qPCR Kit (Sangon, Shanghai, China). GAPDH and U6 were used as an internal reference. The relative RNA levels of ACTA2-AS1, miR143-3p, and SMAD3 were determined by using the formula of 2^−ΔΔCt. The primers in our experiment are as follows: ACTA2-AS1-F: 5′-GTTCTGGAGGCTGGCTTGATATGG-3′, ACTA2-AS1-R: 5′-TCCTTCATCGGTAGGCAACAAACG-3′; SMAD3-F: 5′-CAGCCATGTCGTCCATC-3′, SMAD3-R: 5′-CTCGCACCATTTCTCCTC-3′; miR-143-3P: 5′-CGTGAGATGAAGCACTGTAGCTC-3′; GAPDH-F: 5′-CTCAGACACCATGGGGAAGGTGA-3′, GAPDH-R: 5′-ATGATCTTGAGGCTGTTGTCATA-3′; and U6: 5′-ATTGGAACGATACAGAGAAGATT3′. All assays repeated at least three times.

After RNA isolation from the root of tomatoes, qRT-PCR was performed using SYBR Premix Ex Taq (Takara, Dalian, China) and MicroRNAs qPCR Kit (Sangon Biotech, China), according to the manufacturer’s instructions. Subsequently, qRT-PCR was performed in a 20 μL reaction volume, including 10 μL SYBR Green Master Mix, 0.8 μL PCR Forward Primer (10 μM), 0.8 μL PCR Reverse Primer (10 μM), 0.4 μL ROX, 2 μL cDNA and 6 μL nuclease-free water. The protocol was initiated at 95 °C for 5 min, followed by 95 °C (5 s) and 60 °C (34 s) for a total of 40 cycles. β-actin was used as a reference gene. Reactions were performed in three independent wells. The 2^−ΔΔCt method [41 (link)] was used to calculate the relative RNA expression levels. Student’s t-tests were performed, and results were considered significant when p < 0.05. The values were expressed as means ± standard deviation (SD). The primers sequences are shown in Supplementary Table S1.

To validate the expressions of the miRNAs identified in the sequencing analysis, three miRNAs with different expression patterns in different treatments were randomly selected for qRT-PCR assays. miRNA First Strand cDNA Synthesis (Sangon Biotech, Shanghai, China) was used for polyadenylation and reverse transcription of all miRNAS, according to the manufacturer’s instructions. The MicroRNAs qPCR Kit (Sangon Biotech, Shanghai) was used for miRNA qPCR with the following steps: 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 30 s. All qRT-PCR experiments were performed on the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Soybean U6 snRNA was used as the internal reference gene for the miRNAs, and the data were quantified using the 2^−ΔΔCt method, all the primers used for qRT-PCR were shown in Supplementary Material S5. [69 (link)].