Rotor gene q 2plex hrm system

Total RNA was extracted from the REVec-infected cells using TRIzol (Life Technologies, Grand Island, NY, United States) and reverse transcribed with a Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, United States) using the primers shown in Supplementary Table S1. qRT-PCR assays of REVec genomic and antigenomic RNA were carried out using a gene-specific double fluorescence-labeled probe and THUNDERBIRD Probe qPCR Mix or THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) in a Rotor Gene Q 2plex HRM system (Qiagen, Hilden, Germany). The primers and probe used are shown in Supplementary Table S1.

Total RNA was extracted from cells or tissues by using the UNIQ-10/Trizol total RNA extraction kit (Sangon, #B511361, Shanghai, China) and converted to cDNA using the PrimeScript RT Reagent Kit (Takara, #a4302-1, Otus, Shiga, Japan). The SYBR Green PCR kit (Takara, #aka505, Otus, Shiga, Japan) was used for qRT–PCR. The expression levels of gene mRNA were normalized to the GAPDH levels. The following primer sequences were used: ABCB1 (human) sense 5′-CCCATCATTGCAATAGCAGG-3′ and antisense 5′-TGTTCAAACTTCTGCTCCTGA-3′, EGFR (human) sense 5′-TCTACAACCCCACCACGTAC-3′ and antisense 5′-TTCCGTTACACACTTTGCGG-3′, IL6 (human) sense 5′-AGTCCTGATCCAGTTCCTGC-3′ and antisense 5′-AAGCTGCGCAGAATGAGATG-3′, IL16 (human) sense 5′-AAAACATTTTGCGCGCACAA-3′ and antisense 5′-ACCCAGGCACATCATCAGAA-3′, and GAPDH (human) sense 5′-GGTGGTCTCCTCTGACTTCAACA-3′ and antisense 5′-GTTGCTGTAGCCAAATTCGTTGT-3′.
miRNA was isolated using the mirVana^TM miRNA isolation kit (Ambion, #AM1556, Austin, TX). MicroRNA cDNAs were synthesized using the Taqman® MicroRNA Reverse Transcription Kit (Invitrogen, #4366596). Taqman® MiRNA Assays (Invitrogen, #4427975) were used to amplify the expression of miR-338-5p (ID 002658) and RNU6B (ID 001093) according to the manufacturer’s instructions. The amplification and detection of specific products were performed with the Rotor-Gene Q 2plex HRM System (Qiagen, Valencia, CA, USA).

MEF cells were washed with PBS before performing RNA extraction with the NucleoSpin RNA II Kit (Macherey-Nagel). RNA was quantified with a NanoDrop (Thermo Fisher Scientific) and reverse-transcribed according to the supplier’s protocol (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). Quantitative real-time polymerase chain reactions were performed with KAPA SYBR fast (Kapa Biosystems) and a Rotor-Gene Q 2plex HRM System (Qiagen).