Abscript 2 one step sybr green rt qpcr kit

To minimize selection bias, 3 upregulated and 3 downregulated MMDELs were randomly selected. The expression level of these six MMDELs was quantified using qPCR on a Roche LightCycler 480 (Roche Applied Science). The sequences of the specific primers used and the lengths of the products are indicated in Table I. After the total RNA was isolated from HUVECs according to the aforementioned method. RT-qPCR was performed according to the manufacturer's protocol using a ABScript II cDNA First-Strand Synthesis Kit (cat. no. RK20400; Abclonal Biotech Co., Ltd.) and qPCR was performed using a ABScript II One Step SYBR Green RT-qPCR Kit (cat. no. RK20404; Abclonal Biotech Co., Ltd.). PCR reactions were implemented using the following temperature protocol: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 20 sec. Expression levels of target genes were normalized to that of GAPDH, which used as an internal reference gene. The relative expression of each lncRNA was calculated using the 2^-ΔΔCq method (33 (link)).

According to the Trizol Reagent instructions, extract the total RNA of all tissue samples. Took 1 µl of RNA solution sample, used NanoDrop 2000 spectrophotometer to measure the absorbance value of A260/A280 and measured RNA concentration at the same time. Followed the instructions of ABScript II One Step SYBR Green RT-qPCR Kit (abclonal, China). The 2^−∆∆Ct method was used for analysis. The primer sequences of SKA3 and β-actin were listed in Table 1.Table 1

Sequences of primers.

Table 1

Gene	Forward primer	Reverse primer
SKA3	5´-CAGATCCCTCTTCACCTACGA-3´	5´-TCAACGTTTAAAGGGGGACA-3´
β- actin	5´-GGCATCCTCACCCTGAAGTA-3´	5´-GGGGTGTTGAAGGTCTCAAA-3´