Bulge loop mirna qrt pcr starter kit

Total RNA from the serum, exosomes, cells, or tissues was obtained using Trizol Reagent or Trizol LS Reagent (Invitrogen) according to the manufacturer’s instructions, and each sample was eluted in 30 μl of RNase-free water (Takara, Dalian, China). All reverse transcription of miRNA from maize and Jinhua sow samples was performed using the One Step PrimeScript^®miRNA cDNA Synthesis Kit (Takara) according to manufacturer’s instructions, and qPCR was performed using SsoAdvanced™ SYBR^® Green Supermix (Bio-Rad). Stem-loop qRT-PCR was used to evaluate the miRNA level using the Bulge-Loop^TM miRNA qRT-PCR Starter Kit (RiboBio) according to manufacturer’s instructions, and the reverse transcription and qPCR primers were also synthesized by RiboBio. Meanwhile, mRNA was reverse-transcribed to cDNA using PrimeScript^® 1st Strand cDNA Synthesis Kit (Takara) and qPCR was performed using SYBR^® Premix Ex Taq Kit^TM II (Takara) and Bio-Rad CFX96TM Real-Time PCR Systems (Bio-Rad, Hercules, CA, USA). All reactions were performed in triplicate, and the absolute or relative expression levels of the target miRNAs and mRNAs were calculated as needed.

Total RNA was extracted from cultured GECs by TRIzol reagent (Takara, Otsu, Japan) according to the manufacturer’s instructions. Expression levels of miRNAs were assessed by qRT-PCR using Bulge-Loop TM miRNA qRT-PCR Starter Kit (RiboBio, Guangzhou, China) on ABI 7500 System. The Bulge-LoopTM RT-qPCR primers for miR-590-3p and U6 small nuclear RNA were from RiboBio Company. For mRNAs analysis, cDNA was synthesized with high-capacity cDNA reverse transcription kit (Takara). Quantitative real-time polymerase chain reaction (RT-PCR) was performed with SYBR green qPCR master mix (Takara). The results of real-time PCR were quantified using the 2^−ΔΔCt method (Table 1).

Total RNA was purified from liver or cells using TRIzol (Invitrogen, Carlsbad, CA, USA.) and reverse transcribed to cDNA using TIANScript RT kit (Tiangen Inc., Beijing, China). Subsequently, quantitative PCR amplification was performed using SYBR^TM Select Master Mix on the ABI StepOne Plus Real-Time PCR System (Applied Biosystems). Mir-155 was detected by Bulge-Loop^TM miRNA qRT-PCR Starter Kit (Ribobio, Guangzhou, China). The relative mRNA or miRNA expression levels were calculated by normalizing the level of the target genes against that of the housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the 2^−ΔΔCt method. The specific primer sequences are listed in Table 1.

The cDNA was generated by using BulgeLoopTM miRNA qRT-PCR starter kit (RiboBio Co Guangdong, China) according to the manufacture’s protocol. The reaction was performed in a thermocycler. The reverse transcription process was carried out at 42 ℃ for 60 min and then at 70 ℃ for 10 min. At last, the cDNA was stored at − 20 ℃ until use.

Total RNA was extracted using TRIzol reagent (15596018, Invitrogen) and the cDNA of mRNA was synthesized from 1 μg of total RNA with M-MLV (M170A, Promega, USA). RT-qPCR was performed using a commercial SYBR Green RT-PCR Kit (QPK-201, Takara, Otsu, Japan). The reverse transcription and detection of miRNA was performed using Bulge-Loop^TM miRNA qRT-PCR Starter Kit (C10211, RiboBio Co., Guangzhou, China) and Bulge-Loop^TM hsa-miR-124-5p qRT-PCR Primer Set (miRQ0004591-1-2, RiboBio Co., Guangzhou, China). The detetion of U6 snRNA was used as internal reference (MQP-0202, RiboBio Co., Guangzhou, China). The amplification parameters in this assay as follows: 1 cycle at 95°C for 10 min, 40 cycles of 95°C 15 s and 60°C 1 min. The specific primers of PPP1R13L and GAPDH were constructed by Sangon (Sangon Biotech, Shanghai, China) and their detailed sequences as follows: PPP1R13L (forward: 5′-CAGACAGCGAGCTATGAACG-3′, reverse: 5′-GTGGCGCTAGTGAGGTTGTC-3′); GAPDH (forward: 5′-GGTGGTCTCCTCTGACTTCAACA-3′, reverse: 5′-GTTGCTGTAGCCAAATTCGTTGC-3′). The mRNA and miRNA expression was respectively normalized to the expression of GAPDH and U6, and was expressed as 2^−ΔΔCt after normalization. All samples were assayed in triplicate.