Mirvana mrna isolation kit

Total RNA was isolated from HUVECs infected with adenovirus SREBP2 (Ad-SREBP2) or Ad-null (empty vector). mRNA was isolated using mirVana mRNA isolation kit (Thermo Fisher Scientific). Standard Illumina protocols were used to construct the RNA libraries and sequencing. Analysis was performed using base calling and quality scoring by using Real-Time Analysis version 2 (RTA v2) on the NextSeq 500 system. Data were demultiplexed and converted to FASTQ files using Bcl2fastq conversion software v1.8.4. Sequence reads were trimmed of their adaptor sequences and masked for low complexity or low-quality sequence. Reads were mapped to the h19 genome using tophat v2.0.14 (55 (link)). Data were normalized to reads per kilobase of transcript, per million mapped reads (RPKM) using cufflinks v2.2.1 to assemble transcripts and estimate mRNA abundance (Supplemental Figure 4) (56 (link), 57 (link)). Data are available at NCBI’s Gene Expression Omnibus database (GSE121782).

Total RNA was extracted using the mirVana™ mRNA Isolation Kit (Thermo Fisher) according to the manufacturer's instructions. To quantify the extracted RNA, NanoDrop 1000 (Thermo Fisher Waltham, MA, USA) was used. 800 to 1,000 ng of total RNA was applied to synthetize cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems/Thermo Fisher) according to the manufacturer's instructions. Real time-qPCR reactions were performed using 1 μl of cDNA template, 1 μl of the desired probe, 10 μL of RT-qPCR Master Mix (Applied Biosystems/Thermo Fisher) and nuclease-free water to a final volume of 20 μl. Comparative CT reactions were performed in triplicates using the StepOnePlus™ instrument (Applied Biosystems/Thermo Fisher). Calculations for gene expression changes were performed using the 2^−ΔΔCT method. The human probes used were all from Applied Biosystems/Thermo Fisher and were SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), MMP −2 (Hs01548727_m1), −7 (Hs01042796_m1), and −9 (Hs00957562_m1). ACTB (Hs01060665_g1) was used as control of the reaction amplification.

Total cellular RNA was purified from individual tumors using the mirVANA mRNA isolation kit following manufacturer’s recommendations (Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration and purity of the collected RNAs were assessed using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Model 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA preparations with sufficient mass and integrity (RIN >7.0) were submitted to the Genomics Division of the University of Iowa Institute for Human Genetics for RNA sequencing (RNAseq) [10 (link)]. Total cellular RNA (500 ng) was fragmented, converted to cDNA and ligated to sequencing adaptors containing indexes using the Illumina TruSeq stranded total RNA library preparation kit (Illumina, Inc., San Diego, CA, USA). Molar concentrations of the indexed libraries were measured on the Model 2100 Agilent Bioanalyzer and combined equally into pools for sequencing (Agilent, Santa Clara, CA, USA). The concentration of the pools were measured using the Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and sequenced on the Illumina HiSeq 4000 genome sequencer using a 150 bp paired-end SBS chemistry.