Rnalater rna stabilization reagent

Before surgery, reference biopsies of liver tissue were collected, rinsed with PBS, and placed into RNAlater RNA Stabilization Reagent (Qiagen, Hilden, Germany). The samples were subsequently snap frozen to –80°C and kept at this temperature for 3 months until Rbp4, Ahsg, Fgf21 gene expression analyses were performed.
Eight weeks after surgery, blood for hepatokines' analysis was collected from the abdominal aorta into tubes containing 10 μl of EDTA (Sigma-Aldrich, St. Louis, Mo, USA). After centrifugation at 4 000 rpm for 10 minutes at 4°C, plasma samples were collected and snap frozen in liquid nitrogen and stored at – 80°C until analysis was performed. Hepatokines, such as RBP4, fetuin-A, and FGF21, were assessed in duplicate by ELISA kits (Cloud-Clone Corp., Katy, Tex., USA).
After blood sampling the tissues were harvested and the animals were euthanized. Liver tissue was explanted, rinsed with PBS, and placed into RNAlater RNA Stabilization Reagent (Qiagen, Hilden, Germany). Then samples were snap frozen at –80°C for 1 month until further analysis.

Studies were performed 6 months post-exposure. Given the small size of the irradiated area, different animals were dedicated to histological analyses and mRNA preparations. For histology, the right and left lungs were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer paraffin tissue sections were used for HES and Masson's trichrome staining and for immunohistological studies. For the purposes of mRNA preparation, the irradiated area, ipsilateral and contralateral lung tissues were frozen in an RNAlater^TM RNA Stabilization Reagent (Qiagen, CA) pending analysis. To avoid variations in the structural/cellular constitution of different areas of the lung, age-matched control/unirradiated mice were included for lung imaging and histological analyses and measurements were performed in matched areas between irradiated and non-irradiated mice.

Two flasks of HUVECs from same passage were cultured with NG-ECGM and HG-ECGM for 72 h, respectively. Subsequently, HUVECs were harvested and transferred into RNAlater^TM RNA Stabilization Reagent (Qiagen, Hilden, Germany) for homogenization. MiRNAs were isolated using the Allprep RNA isolation kit (Qiagen) according to the manufacturer’s protocol. The RNA integrity number (RIN) was determined using the Agilent Bioanalyzer 2100 Expert (B.02.08.SI648, Agilent, Santa Clara, CA, USA). The RNA samples of HG and NG with RINs ranging from 7 to 10 were sent to the Beijing Genomics Institute (BGI, Shenzhen, China) to perform the miRNA sequencing. Filtered miRNAs were quantified by realigning reads to predicted miRNAs in QuickMIRSeq [15 (link)]. To decrease false positives, the data were extensively filtered by joint mapping to the transcriptome and ribosomal RNA using QuickMIRSeq. Sequences were aligned to the reference genome GRCh38.p13. The count data were transformed to log2-counts per million (logCPM) using the voom function from the limma package [16 (link)] in R. Differential expression analysis was performed utilizing the limma package. A false positive rate of α = 0.05 with a false discovery rate (FDR) correction was considered as the level of significance. Volcano plots and heatmaps were created using ggplot2 package (version 2.2.1) and the iDEP [17 (link)](version 0.91).