Illumina HiSeq 2000 sequencing; MicroRNA expression profile of methamphetamine-sensitized and saline-control mice examined by high throughput sequencing
MicroRNA expression profile of methamphetamine-sensitized and saline-control mice examined by high throughput sequencing
Mice (male, 20-25g) were given once daily injections of saline for two consecutive days (day 1-2), after which they were divided into two groups randomly. Two groups of mice were then given once daily injections of methamphetamine (METH, 2mg/kg) or saline for five consecutive days (day 3-7) followed by two injection-free days (day 8-9). On day 10, mice were given a challenge injection of either 2 mg/kg dose of METH or saline. Horizontal locomotor activity was performed on all drug treatment days for 60 minutes before and after the injections. For all experiments, mice were sacrificed 24 h after the last injection. After mice were sacrificed, nucleus accumbens (NAc) were harvested and NAc lysate from eight mice of each group were pooled as one sample for total RNA isolation, following to the manufacturers instructions of TRIzol (Invitrogen, USA). Two RNA samples from each group were prepared and designated saline 1, saline 2, METH 1 and METH 2. The RNA quality was evaluated by Agilent 2100 BioAnalyzer (Agilent Technologies, USA), and all samples exhibited an RIN greater than 8. 10ug of total RNA per sample were electrophoresed through a 15% TBE urea polyacrylamide gel. The 18- to 30-nt fraction was excised and recovered. The isolated small RNAs were then ligated to 5' and 3' adaptors with RNA ligase. Small RNAs were gel-purified after each ligation and were then reverse transcribed. The resulting cDNAs were PCR-amplified using Illumina primers, and the PCR products were then purified on a nondenaturing acrylamide gel.