5 protocols
nucleic acid sequencing
NextGen sequencing libraries were prepared using small RNA sample preparation kits from Illumina (San Diego, CA). The 3 and 5 adapters and the reverse transcription primer were diluted in nuclease-free water to concentrations specified by Illumina. RNA isolated from 100 l of plasma was concentrated and mixed with the diluted 3 adapter in a final volume of 6 l of nuclease free water. Blast was used to search miRNA (miRBase) and Bowtie was used to search other large databases (mm9, NCBI nr)
The mice were euthanized at different time points post exposure (at 3, 8, 24, 48 and 72 hours);
nucleic acid library construction protocol
we constructed a single library with an insert size ranging from 20 to 100 nucleotides long. We also did not conduct mRNA purification and fragmentation steps prior to library construction since we did not observe ribosomal RNA peaks from plasma RNA on the bioanalyzer, and most of the transcripts in circulation were probably not full-length based on prior studies. This approach allowed us to obtain a general profile for both miRNA and mRNA in the plasma from a single library.
Eight week old female C57 Black mice were fasted for 24 hours prior to a single intraperitoneal injection of 350mg/kg of acetaminophen in phosphate buffer saline (PBS) (treatment group) or PBS (control group)
nucleic acid extraction
Liver and plasma samples were collected from different time points after mice had been exposed to acetaminophen. The plasma was prepared from EDTA blood by centrifugation at 1000 x g for 15 minutes to separate the plasma and blood cells. Total RNA from liver and plasma was extracted with the miRNeasy kit (Qiagen, Germantown, MD), as described. The quality and quantity of the RNA were evaluated with Agilent 2100 Bioanalyzer (Santa Clara, CA) and NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE).