Sybr primscript rt pcr kit

Expressions of IP, TP receptors, and β-actin (internal controls) were detected by real-time PCR. Vessel specimens (pooled from 2 mice for each single set of experiments) were cut open and rinsed of blood components, followed by mincing and homogenizing in an ice-cold RNAiso Plus solution (TaKaRa, Dalian, China), using a glass homogenizer. In some experiments, the opened vessel strips were further denuded of endothelium by rubbing with a moistened cotton swab, which was made around one-tip of a micro-dissecting forceps, under a binocular microscope. Total RNA was prepared according to the manufacturer’s instructions. First-strand cDNA was synthesized using total RNA (250 ng) and oligo(dT)15 primers (TaKaRa). The PCR primers for IP, TP receptors, and β-actin were described previously (19). Real-time PCR was performed using a SYBR PrimScript RT-PCR kit (TaKaRa).

The total RNA in cells and tissues was extracted by Trizol (Invitrogen). The RNA samples of A206/A280 from 1.8 to 2.0 were reversely transcribed. Complementary DNA was synthesized in accordance with reverse transcription PCR with RNA as a template. qPCR was performed with SYBR PrimScript RT-PCR Kit (Takara Bio Inc., Otsu, Shiga, Japan). The target gene expression was analyzed by 2^−ΔΔct method. The primers were synthesized by Invitrogen (Table 1).
Table 1

Primer sequence

Gene	Sequence
FOXP4-AS1	F: 5′-GTGAGCTTCTGGGTTCGACA-3′
	R: 5′-ATTGAGGGTTAGGGCAGCAC-3′
EZH2	F: 5′-AATCAGAGTACATGCGACT GAGA-3′
	R: 5′- GCTGTATCCTTCGCTGTTTCC-3′
ZC3H12D	F: 5′-AAATATAGTTTGTAGGAGGAAGAGTGTTA-3′
	R: 5′-CAATAAAAAACCACAAAACCATATTATCTC-3′
GAPDH	F: 5′- CACCCACTCCTCCACCTTTG -3′
	R: 5′- CCACCACCCTGTTGCTGTAG-3′

F, forward; R, reverse; FOXP4-AS1, forkhead box P4 antisense RNA 1; EZH2, enhancer of zeste homolog 2; ZC3H12D, zinc finger CCCH-type containing 12D; GAPDH, glyceraldehyde phosphate dehydrogenase