Rt qpcr primers

Total RNA from tissues and cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA concentration was estimated using a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). The RT-qPCR primers were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). cDNA was synthesized from total RNA and the relative mRNA and non-coding RNA levels were determined using a RT kit (Toyobo Life Science). The expression levels of miR-29a-3p were determined by normalization to U6 small nuclear RNA by the 2^−ΔΔCq method (34 (link)). An ABI 7500 Sequence Detection System (Thermo Fisher Scientific, Inc.) and SYBR-Green Real-Time PCR Master Mix (Toyobo Life Science) were used to perform RT-qPCR according to the manufacturer's instructions. A total of 20 µl PCR reaction mixture consisting of 1 µl reverse primers, 1 µl forward primers, 10 µl SYBR-Green Master Mix, 6 µl DEPC and 2 µl synthesized cDNA was used. GAPDH was used as an internal control. RT was performed as follows: 25°C for 10 min, 37°C for 120 min, 85°C for 15 min, then 4°C. PCR conditions were as follows: 94°C for 5–10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 45 sec and final extension at 72°C for 2–3 min. LASP1 expression levels were normalized to those of GAPDH using the 2^−ΔΔCq method. The primer sequences are listed in Table I.