Express sybr greener mirna qrt pcr kit

For mRNA detection, the TaqMan Reverse Transcription Kit (Life technologies) was used to reverse-transcribe RNA according to the manufacturer protocol. qPCR was performed using Taqman Fast Advanced Universal PCR Master Mix (Life Technologies), and reactions were analyzed using a 7500 Fast Real-Time PCR Instrument (Applied Biosystems). The complete list of Taqman assay IDs are provided in S3 Table. Data analysis was performed using the comparative C_T method, and statistical significance was determined using a two-tailed Student’s t-test (p<0.05). For validation experiments, a distinct RNA samples from that used for microarray analysis was generated to ensure reproducibility of results.
For mature miRNA detection, poly(A) tailing and reverse transcription of total RNA was conducted using the NCode VILO miRNA cDNA Synthesis Kit (Invitrogen) according to the manufacturer protocol. qPCR was performed using the Express Sybr Green ER miRNA qRT-PCR Kit (Invitrogen), and reactions were analyzed using a 7500 Fast Real-Time PCR Instrument (Applied Biosystems). Primers used for specific RNA detection included U6 5′-GCAAATTCGTGAAGCGTTCCAT, miR-21 5′-CGGTAGCTTATCAGACTGATGTTGA, miR-183 5′-GGTATGGCACTGGTAGAATTCACT, miR-96 5′-TGGCACTAGCACATTTTTGCT, miR-182 5′-CTTGGCAATGGTACAACTCACA.

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. After digestion with DNase and cleanup, RNA samples were quantified, aliquoted, and stored at −80°C. Total RNA isolated from tissue samples and/or cultured cells were used as a template to synthesize cDNA for quantitative RT-PCR (qRT-PCR) analysis in a StepOne Real-time PCR system (Applied Biosystems, Foster City, CA, USA).
The differential miRNA expression patterns were validated with the TaqMan qRT-PCR assay (Applied Biosystems) or the NCode VILO miRNA cDNA Synthesis kit for qRT-PCR and EXPRESS SYBR GreenER miRNA qRT-PCR kit (Invitrogen). qRT-PCR with SYBR Green dye (Applied Biosystems) was used to assess mRNA expression. RNU48 (for TaqMan qRT-PCR) or 5.8S (for SYBR qRT-PCR) and GAPDH were used as endogenous controls for qRT-PCR of miRNA and mRNA, respectively. Each sample was run in triplicate.

Total RNA was isolated using a UNIQ-10/Trizol total RNA extraction kit (Sangon, Shanghai, China) and was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Otus, Shiga, Japan). The primer sets used are listed in Supplementary Table 1. Quantitative real-time RT-PCR (qRT-PCR) analysis was performed using SYBR Premix Ex Taq (Takara).
miRNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. RT and miRNA detection were conducted using the NCode VILO miRNA cDNA Synthesis Kit and the EXPRESS SYBR GreenER miRNA qRT-PCR Kit, respectively (Invitrogen, Carlsbad, CA, USA). miRNA-specific forward primers were designed according to the manufacturer's instructions; these primers are also listed in Supplementary Table 1. The observed miRNA expression levels were normalized to the U6 expression levels.

Total RNA samples and miRNAs of human plasma, SH-SY5Y cells, and brain tissues were extracted by TRIzol reagent (Life Technologies, Rainbio, MA, USA) and miRNeasy Mini Kit (Qiagen) in accordance with the manufacturer's manual, respectively. The purity of RNA was checked at the absorbance of 260/280 and 260/230 with the Ultra-micro UV Spectrophotometer (Beckman Coulter, CA, USA). For cDNA synthesis of MafB, iNOS, and COX-2, 1 μg total RNAs was reverse-transcribed using the EasyScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd., Beijing, China) and the RT-PCR was performed via SYBR Premix Ex Taq (TaKaRa, Shiga, Japan). For detection of miR-155, cDNA was constructed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, CA, USA), and RT-PCR reactions were implemented via the Express SYBR GreenER miRNA qRT-PCR kit (Invitrogen, Rainbio, MA, USA). All amplification assays were performed on a Bio-Rad Real-Time PCR equipment (Bio-Rad, Hercules, CA) based on the standard protocol. The expression levels of MafB, iNOS, and COX-2 were standardized to GAPDH, and miR-155 was normalized against U6 expression. The correlative quantification analysis of target genes was detected by comparing to the internal reference using formula 2^−∆∆Ct, where ∆Ct = Ct_miR−XorX − Ct_{U6 or GAPDH}.

Total RNA was extracted from the cells using the TRIzol reagent (15596018, Thermo Fisher Scientific, USA), and the concentration of the RNA was detected by using Nanodrop (FSC-6539918, eGeneralMedical.com, USA) and diluted to 500 ng/μL. For miRNAs, NCode VILO miRNA cDNA Synthesis and EXPRESS SYBR GreenER miRNA qRT-PCR Kits (Life Technologies) were applied for detecting the expression levels. For mRNAs, total RNA (1 μg) was converted into cDNAs using a Superscript II first-strand cDNA synthesis system (Invitrogen, USA). The mRNA expression levels were determined by SYBR-Green PCR Master Mix (Thermo Fisher Scientific, USA) in the 7500 Real-Time PCR system (Thermo Fisher Scientific, USA). Conditions of the PCR cycle were set as follows: pretreatment at 95°C for 30 s, followed by 60°C for 30 s and 60°C for 30 s for 45 cycles. The 2^−ΔΔCT method was used to determine the expression levels of RT-PCR products (Livak and Schmittgen [24 (link)]). All primer sequences used are listed in Table 1.

Total RNA was isolated from the cell lines using TRIzol (Life Technologies) and quantified. Equal amount of RNA was used for the one-step or two-step qPCR performed using the Superscript III SYBR Green qRT-PCR kits, according to manufacturer’s instructions (Life Technologies). For miRNA, PCR was performed using NCode VILO miRNA cDNA Synthesis and EXPRESS SYBR GreenER miRNA qRT-PCR Kits (Life Technologies), according to the manufacturer’s protocol. The primers (sequences provided in the Supplementary materials and methods; Additional file 7) were designed using Primer 3 [48 (link)] and synthesized by Integrated DNA Technologies (Coralville, IA). PCR was performed using Realplex² Mastercycler ep gradient S thermal cycler (Eppendorf).