Premix extaq quantitative pcr kit

TRIzol reagent (Gibco) was applied for total RNA isolation. The PrimeScript RT Kit (TaKaRa, Tokyo, Japan) was used to synthesize cDNA. The mRNA expression level was studied using a Synergy (SYBR) Premix ExTaq Quantitative PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA) and LightCycler instrument (Roche, Basel, Switzerland). U6 and GAPDH served as internal controls. The relative expressions of miRNA-181b-5p and SSX2IP were quantified by 2^-ΔΔCt method. The primers for qRT-PCR were acquired from Shanghai Biotechnology Co., Ltd. (Shanghai, China). The primers used were as follows: miRNA-181b-5p: forward primer 5’-CCAGCTGGGCTCACTGAACAATGA-3’, reverse primer 5’-CAACTGGTGTCGTGGAGTCGGC-3’; U6: forward primer 5’-CTCGCTTCGGCAGCACA-3’, reverse primer 5’-AACGCTTCACGAATTTGCGT-3’; SSX2IP: forward primer 5’ CCGGGGAACTAAGCAGAGAGA-3’, reverse primer 5’-GTTCATGGTCTTGTCGTGAGAT-3’; GAPDH: forward primer 5’-GCACCGTCAAGCTGAGAAC-3’, reverse primer 5’-TGGTGAAGACGCCAGTGGA-3’.

The Trizol kit (Ambion) was employed for total RNA isolation. PrimeScript RT reagent kit (TaKaRa) was applied to synthesize complementary DNA (cDNA). Synergy Brands (SYBR) Premix ExTaq quantitative PCR kit (Thermo Fisher Scientific) and Light Cycler instrument (Roche, Switzerland) examined the levels of miR‐199b‐3p and AURKA mRNA. AURKA used GAPDH while miR‐199b‐3p applied U6 as an internal control. The 2^−ΔΔCt value was applied to compare relative expression differences. The primer sequences are as given in Table 1.

The total RNA was extracted using Trizol reagent (Gibco). Then complementary DNA (cDNA) was synthesized with the PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan). Expression levels of mRNAs were investigated with the Synergy Brands (SYBR) Premix ExTaq quantitative PCR kit (Thermo Fisher Scientific, Lafayette, Colorado, USA) and Light Cycler instrument (Roche, Basel, Switzerland). U6 expression was then performed as an endogenous control for miR-490-3p normalization, while GAPDH was used for an internal reference for AURKA normalization. The relative expression of miR-490-3p and AURKA was quantified using the 2^–ΔΔCt method. Table I shows primers used in quantitative real-time polymerase chain reaction (qRT-PCR), which were synthesized by Shanghai Biotechnology (Shanghai, China).