Oligo dt primer

LN229, U87MG, GBM8401, U118MG, and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin, and streptomycin. Total RNA was extracted using the EasyPure Total RNA reagent (Bioman, Taipei, Taiwan) according to the manufacturer’s protocol. For cDNA synthesis, 1.0 μg RNA was reverse transcribed (RT) into cDNA using Oligo dT primer with MMLV Reverse Transcriptase (Epicentre Biotechnologies, Madison, WI, USA). The normal brain cDNA was purchased from Origene Technologies (Rockville, MD, USA).
Gene expression was quantified by qRT-PCR and performed in illumina ECO™ Real-Time PCR system. The amplifications were done using IQ² fast qPCR system with ROX (Bio-genesis Technology Inc., Taipei, Taiwan). The relative quantitative gene expression against an internal control, GAPDH was performed using the 2^−ΔΔCt method. The primer pairs used were: DDX3X forward, 5′-ATGGCTTGTGCCCAAACAG-3′ and reverse, 5′-CGCCTGGACCATCTGAATAAA-3′ [28 (link)] and GAPDH forward, 5′-CTTCATTGACCTCAACTAC-3′and reverse, 5′-GCCATCCACAGTCTTCTG-3′.

Total RNA was extracted using EasyPure Total RNA reagent (Bioman, Taipei, Taiwan) according to the manufacturer’s protocol. For cDNA synthesis, 1.0 μg RNA was reverse transcribed into cDNA using Oligo dT primer with MMLV Reverse Transcriptase (Epicentre Biotechnologies, Madison, WI, USA). Normal brain cDNA was purchased from Origene Technologies (Rockville, MD, USA).
Gene expression was quantified using quantitative RT-PCR (qRT-PCR) and performed in an illumina ECOTM Real-Time PCR system. Amplifications were performed using an IQ2 fast qPCR system with ROX (Bio-genesis Technology Inc., Taipei, Taiwan). Relative quantitative gene expression against an internal control, GAPDH, was performed using the 2^−ΔΔCt method^{22 (link)}. The primer pairs used were SGO2 forward, 5′-ATGTGGTGCATGGCCTAAAAA-3′ and reverse, 5′-GGGGTACATATTGGTGATCTGC-3′ and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-ATGGTGGTGAAGACGCCAGT-3′.

First-strand cDNA were reversely transcribed from mRNA templates of each skin sample in equal amount, using MMLV High Performance Reverse Transcriptase Kit with an oligo(dT) primer (EPICENTRE Biotechnologies, Madison, WI). Then, MHC-I related target gene expression was quantified by Bio-RAD iQ5 real-time PCR detection system, using Maxima SYBR Green/ROX qPCR Master Mix (2X) (Fermentas, Thermo Scientific). The specificity of real-time PCR reaction was further confirmed by melting curve analysis. Normalized values for target mRNA expression in each sample were calculated as the relative quantity of target gene divided by the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the gene primers were designed by online primer design tool of Primer 3 as shown in Table 1.