Bμlge loop mirna qrt pcr primer

Total RNA was extracted from tissues or cells using Trizol reagent (TaKaRa, Japan) according to the manufacturer’s instructions. The integrity and concentration of all obtained RNA samples were assayed by 1.5% agarose gel electrophoresis, and determined by measuring the optical density in a Nanodrop 2000c spectrophotometer (Thermo, Waltham, MA, USA) at 260/280 nm ratio.
cDNA synthesis for mRNA was carried out using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan), which was able to eliminate genomic DNA. For miRNA, BμLge-Loop™ miRNA qRT-PCR Primer specific for gga-miR-16-5p and U6 were designed by RiboBio (RiboBio, Guangzhou, China), and ReverTra Ace qPCR RT Kit (Toyobo, Japan) was used to synthesize cDNA.
The RT-qPCR reactions were carried out in a Bio-Rad CFX96 Real-Time Detection instrument (Bio-Rad, Hercules, CA, USA). The iTaq Universal SYBR Green Supermix Kit (Toyobo, Japan) was used for cDNA quantification, according to the indicated manufacturer’s protocol. Chicken β-actin and U6 were used as internal controls. As described previously, data analysis was carried out with the comparative 2^−ΔΔCT method^{43 (link)}.

Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) as recommended by the supplier. The integrity and quantity of RNA were assessed using 1.5% agarose gel electrophoresis and a spectrophotometer (ND-2000, NanoDrop Inc., Wilmington, DE, USA).
Complementary RNA (cDNA) synthesis for messenger RNA (mRNA) was carried out using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Otsu, Japan). For miRNA, BμLge-Loop™ miRNA qRT-PCR Primer specific for miR-1611 and U6 were designed by RiboBio (RiboBio, Guangzhou, China), and a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) was used to synthesize cDNA.
A real-time qPCR with iTaq Universal SYBR Green Supermix Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used, and analysis was performed with the 2^−ΔΔCt method, as described previously [29 (link)]. Chicken β-actin and U6 were used as internal controls. Primers for RT-qPCR are shown in Table S1.