Oligo dt

Total RNA was extracted from cells using TRIzol^® reagent (cat. no. 3101-100; Shanghai Pufei Biotechnology Co., Ltd.). Total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (cat. no. M1705; Promega Corporation). The Oligo dT was provided by Sangon Biotech Co., Ltd. (cat. no. B0205) and the dNTPs (10 mM) were provided by Promega Corporation (cat. no. U1240). After heat treatment of RNA/primer mixture at 70°C for 10 min and cooling immediately on ice for 10 min, the RT reaction was processed at 42°C for 1 h and 70°C for 10 min. Subsequently, qPCR was performed using ExTaq (Takara Bio, Inc.). The SYBR Master Mixture was provided by Takara Bio, Inc. (cat. no. DRR041B). The following primer were used for the qPCR: RRBP1 forward, 5′-GAGATGGCGAAAACTCACCAC-3′ and reverse, 5′-CTCGAAGGAGGACAGTCACAT-3′; CCR7 forward, 5′-TTCATCGGCGTCAAGTTCC-3′ and reverse, 5′-AAGGTGGTGGTGGTCTCG-3′; and GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 30 sec; followed by 40 cycles of 95°C for 5 sec; and final extension at 60°C for 30 sec. mRNA expression levels were quantified using the 2^−ΔΔCq method (17 (link)) and normalized to the internal reference gene GAPDH.

Total RNA was extracted from KIF3A-siRNA or NC-siRNA transfected cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, and the purity and concentration of RNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription of the RNA was performed using Moloney Murine Leukemia Virus Reverse Transcriptase (Ambion; Thermo Fisher Scientific, Inc.) and a mixture of anchored Oligo(-dT) (Sangon Biotech Co., Ltd.) and dNTPs (Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: 37°C for 50 min, 70°C for 15 min, 4°C for 5 min. The qPCR (SYBR-Green I) was subsequently performed using UltraSYBR Mixture (CoMin Biosciences) according to the manufacturer's instructions. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 5 min; 40 (two-step) cycles at 95°C for 10 sec and 60°C for 35 sec. Relative expression levels were calculated using the 2^−ΔΔCq method and normalized to the internal reference gene β-actin (13 (link)). The following primer sequences were used for qPCR: KIF3A forward, 5′-AGGAGAGTCTGCGTCAGTCT-3′ and reverse, 5′-TTTCAGGCTTTGCAGAACGC-3′; IFT57 forward, 5′-ATGGCGGAGTAACTGAACGG-3′ and reverse, 5′-ATCTTCACCAAAGGAGTGCCG-3′; and β-actin forward, 5′-CTCTTCCAGCCTTCCTTCCT-3′ and reverse, 5′-AGCACTGTGTTGGCGTACAG-3′).