Hifair 3 1st strand cdna synthesis kit with gdna digester plus

Total RNA was extracted from treated PC3M cells using TRIeasyTM Total RNA Extraction Reagent (YEASEN, Shanghai, China) and the mRNA was reversely transcribed into stable cDNA with Hifair^® III 1st Strand cDNA Synthesis Kit with gDNA digester plus (YEASEN, Shanghai, China). RT-qPCR analysis was performed by using the Hieff^® qPCR SYBR Green Master Mix (YEASEN, Shanghai, China). The thermos cycling conditions included: 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 10 s and 60 °C for 30 s. GAPDH was used as an endogenous housekeeping gene. The threshold cycle (CT) was calculated in accordance with the 2^−∆∆Ct method relative to the expression of GAPDH. All the primer sequences are listed below: HIF-1α (forward primer: 5′ATCCATGTGACCATGAGGAAATG-3′, reverse primer: 5′-TCG GCT AGT TAG GGT ACA CTT C-3′), GAPDH (forward primer: 5′-CCA AGG CTG TGG GCA AGG-3′, reverse primer: 5′-GCT CAG TGT AGC CCA GGA TG-3′).

The MolPure Cell RNA Kit (Yeasen, China) was chosen for the extraction and purification of RNA from NSCLC tissues and cells. RNAs shorter than 200 nucleotides were extracted with MolPure Cell/Tissue miRNA Kit (Yeasen, China). The reverse transcription was executed using the Hifair III 1st-Strand cDNA Synthesis Kit with gDNA digester plus (Yeasen, China). To evaluate the expression levels of LDLRAD3, miR-20a-5p, and SLC7A5, the qRT-PCR process was achieved with the Q960 PCR instrument (Dinai, China) using the PC46-THERMOscript SYBR Green qRT-PCR Kit (Aidlab, China). The primer sequences were as follows: LDLRAD3 forward: 5′-CTT GCT GGA CCA GAG AAC ACA TG-3′, reverse: 5′-CAT GAG GTT GTT CCG CTT CCC A-3′; miR-20a-5p forward: 5′-UAC AGC GCA GAC AGU GCA GCU AG-3′, reverse: 5′-CUA GCU GAA CUA CGC ACU GUA-3′; SLC7A5 forward: 5′-GAA GTC ACC AAG TAC ACT GGA TGT-3′, reverse: 5′-GAA GTA GTC CAG GTT GGT CAG A-3′; U6 forward: 5′-GGA AGC TTG TCA TCA ATG GAT ATC-3′, reverse: 5′-TGA TGA CCC TTT TGG CTC CAA C-3′; and β-actin forward: 5′-CAT TGT TAC CAA CTG GGA CGA CAT-3′, reverse: 5′-GCC TCG GTG AGC AGC TTA CA-3′. The relative expression levels of LDLRAD3 and SLC7A5 mRNA were normalized with β-actin, and the relative expression level of miR-20a-5p was normalized with U6 via the 2^−ΔΔCt method.