Cdna kits

Total RNA was isolated from PA-1 cells using TRIzol reagent method as per instructions given by manufacturer (Thermo Fisher Scientific, Inc.). Next, cDNA was constructed using collected RNA samples by cDNA kits according to the manufacturer’s protocol (Thermo Fisher Scientific, Inc.). The RT-PCR was carried out with the help of SYBR GREEN PCR master mix kit (Invitrogen, Karlsruhe, Germany) using Applied Biosystems Real-Time PCR System. The following sets of primers were used for RT-PCR. The samples were run in triplicates and for expression fold changes, Ct values were first normalized within the sample with housekeeping gene i.e., β-actin and then the relative mRNA expression was quantified by the 2-ΔΔCt technique.

Total RNA from Calu-3 and NCI-H661 cells or tumor tissues was acquired using TRIzol^® (Thermo Fisher Scientific, Inc.), and the purity was detected. For miR-106a-5p, complementary DNA was synthesized using a miScript kit (Qiagen GmbH) and a miScript SYBR^® Green PCR kit was used for qPCR (60 min at 42˚C, 5 min at 70˚C; then held at 4˚C). For mRNA detection, qPCR (95˚C for 10 sec, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 1 min) was performed using cDNA kits (Thermo Fisher Scientific, Inc.) and SYBR^® Green PCR Master Mix (Roche Diagnostics), respectively. The standardized reference genes were U6 and GAPDH. The 2^-ΔΔCq method was used to analyze the relative expression levels of target miRNA and genes (32 (link)). The primer sequences are presented in Table I.