Bulge loop mirna qrt pcr kit

To analyze miR-219 expression, cells were transfected with either miR-219 mimic or mimic control for 48 hours using a riboFECT™ CP transfection kit according to the manufacturer’s protocol (RiboBio, Guangzhou, China) before extracting total RNA from neurons using a mirVana miRNA Isolation kit (Thermo Fisher, Waltham, MA, USA). To analyze CaMKIIγ mRNA expression, total RNA was extracted from neurons using Trizol reagent (Invitrogen). A NanoDropND-1000 spectrophotometer (NanoDrop Tech, Wilmington, DE, USA) was used to measure RNA concentrations. One μg of RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China).
Expression of miR-219 was examined using qRT-PCR with a Bulge-Loop miRNA qRT-PCR kit (RiboBio) and miR-219–specific primers. qRT-PCR parameters were as follows: 95°C for 10 seconds, 40 cycles of 95°C for 5 seconds and 60°C for 20 seconds. Primers used for qRT-PCR (synthesized by Sangon Biotech, Shanghai, China) of CaMKIIγ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are listed in Table 1. The experiment was repeated in triplicate. Expression of miR-219 was calculated as relative expression to internal reference U6, while expression of CaMKIIγ mRNA was calculated as relative expression to internal reference GAPDH. The 2^−ΔΔCt method was utilized to analyze data (Yu et al., 2012).

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA from cells, according to the manufacturer's protocol. The PrimeScript RT Reagent kit (Takara Bio, Inc.) was used to convert RNA into cDNA. The condition of RT was 15 min at 37°C and 5 sec at 85°C. The miRNA Extraction kit (Guangzhou RiboBio Co., Ltd.) was used to convert miRNA into cDNA. Quantification of the transcript expression levels were obtained by performing qPCR using SYBR Premix Ex Taq (Takara Bio, Inc.). The ABI StepOne Plus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for qPCR using the SYBR-Green PCR kit (Takara Bio, Inc.). The qPCR protocol consists of the following steps: i) Initial denaturation for 5 min at 95°C; and ii) 40 cycles involving denaturation at 95°C for 10 sec, followed by annealing and extension at 60°C for 34 sec. Each experiment was repeated three times. The miR-22 primers used were from the Bulgeloop miRNA qRT-PCR kit (Guangzhou RiboBio Co., Ltd.). Detailed information of the primers employed for the qPCR experiments is presented in Table I. The 2^−ΔΔCq analysis method was used as quantification method (29 (link)).

Quantitative RT-PCR was conducted as described previously.28 The primers used for GAPDH, Arg1 (The result of agarose gel electrophoresis for ARG1 was shown in Supplementary Figure 1), iNOS and IL1B are listed in Supplementary Table 1. To analyze miRNA expression, we used miDETECT A Track miRNA qRT-PCR Kit and a Bulge-Loop miRNA qRT-PCR Kit (RiboBio, Guangzhou, China) for reverse-transcribed, according to the manufacturer’s introduction. Expression levels of miRNA were normalized to those of U6-snRNA expression while mRNA of IL-10, IL1B and iNOS were normalized to GAPDH expression. All experiments were performed in triplicate.