RT-qPCR
Cells were grown for 48 hours after lipofection before being harvested
using RNAprotect (Qiagen). Total RNA was extracted using Direct-zol RNA
microprep kit and quantified. Each condition was performed in triplicate
using 50 ng of total RNA. For each 20 ul reaction, RNA was added to Luna
Universal One-Step RT-qPCR master mix (New England Bio Labs) and two
RT-qPCR primers at a final concentration of 0.4 µM. The following
amplification cycle was run on a QuantStudio 3 real-time PCR machine
(ThermoFisher Scientific): 55 °C for 10 min, 95 °C for 1 min, followed
by 95 °C for 10 sec and 60 °C for 1 min for 40 cycles. All genes were
normalized to beta Actin (ACTB) and expression was determined via
relative quantification to lipofectamine only carrier control. Gene
specific primers for each gene of interest were ordered from Integrated
DNA Technologies and are as follows: ACTB: 5’-CACCATTGGCAATGAGCGGTTC-3’
and 5’-AGGTCTTTGCGGATGTCCACGT-3’, TBK1: 5’-GGATCACTGCCATTTAGACCC-3’ and
5’-CAGGCATGTCTCCACTCCAG-3’ (PrimerBank 309747068c3), KRT14: 5’-
TGAGCCGCATTCTGAACGAG-3’ and 5’-GATGACTGCGATCCAGAGGA-3’ (PrimerBank
83641893c1), HNRNPA1: 5’-TCAGAGTCTCCTAAAGAGCCC-3’ and
5-ACCTTGTGTGGCCTTGCAT-3’ (PrimerBank 83641893c1).