Total rna 6000 nano kit

Blood samples were collected in the quarantine nurseryat ~ 27 days of age, using Tempus Blood RNA Tubes (Thermo Fisher Scientific, USA) and then stored at − 80 °C until RNA extraction. The RNAs were isolated using Preserved Blood RNA Purification Kit I (Norgen, Canada) according to the manufacturer’s instructions. The RNA integrity number (RIN) of each extracted RNA was assessed by the 2100 Bioanalyzer (Agilent Technologies, USA) using the Eukaryote total RNA 6000 Nano kit. The RIN score was on average 7.9 and ranged from 4.1 to 9.9 (Table 2). WBC differentials were quantified on whole blood samples in K2 ethylenediaminetetraacetic acid (EDTA) tubes (Thermo Fisher Scientific, USA) taken at the same time, using the flow cytometry-based hematology analyzer (ADVIA®2120i Hematology System, Simens Healthineers, Germany) according to the manufacturer’s instructions [59 (link)]. The log2 transformed proportion of each WBC type was used to adjust gene expressions levels for blood cell composition (see later).

For the microarray analysis, total RNA was extracted from tumors of mice (n = 3) from three groups (DC alone, DC + TL, and DC + TL + TRF). Total RNA was also extracted from 4T1 cells for this analysis. Total RNA was extracted using the TRI-reagent solution according to the manufacturer’s instructions (Molecular Research Center, Inc., Cincinnati, OH, USA). The concentrations of the extracted RNA and ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio) were measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity of the extracted RNA samples was evaluated with the RNA integrity number (RIN) for each sample using the Total RNA 6000 Nano Kit with the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The RIN describes a gradual scale of RNA integrity from 1 (RNA completely degraded) to 10 (RNA without degradation). In general, a RIN that is higher than seven is accepted to be optimal in most of experiments. (http://www.biomedicalgenomics.org/The_RNA_Integrity_Number.html)

Subcutaneous tissue samples (including subcutaneous muscle, <500 mg) were excided under deep general anesthesia, and immediately dipped in liquid nitrogen for 1 minute then moved and maintained at −80 °C until use. Total RNA was extracted and purified from dry frozen tissue quickly dipped in Trizol (Cat#15596-026, Invitrogen, USA) homogenized with a 5 mm blade tip (IKA, DE) and processed according total RNA extraction protocol. The RNA obtained was quantified by Nanodrop 2100 and tested for standard quality parameters (RIN, RNA Integrity Number) through Total RNA 6000 Nano kit (Cat#5067-1511, Agilent, DE). The RIN qualified RNA samples were sent to Illumina Sequencing Services of Partner Institute for Computational Biology Omics Core and cDNA libraries were constructed for sequencing as described in Illumina TruSeq™ RNA sample preparation v2 guide (Catalog # RS-930–1021). Sequencing was performed by Illumina HiSeq 2000.