MicroRNA expression profiling of individual rat hypothalamic nuclei
Instrument: Precellys® 24 Dual with Cryolys cooling device
Sample: Hypothalamic tissue (frozen)
Protocol:
- Precellys kits: 03961-1-003 (CK14).
- Sample: ~1-3 mm3 of frozen hypothalamic tissue.
- Extraction solvent: 700μl QIAzol lysis reagent (Qiagen).
- Run the Precellys 24 at 5500 rpm, 1x20 sec.
- Small RNAs were purified using the miRNeasy Mini Kit (Qiagen) with the two-column system.
- Small RNAs were recovered in a volume of 30μL of RNase-free H2O and size-fractionated on a denaturing urea (8M) polyacrylamide (17%) gel.
- RNAs of 16-30 bases were eluted in 0.4M NaCl by overnight incubation under gentle shaking at +4ô€?²C, then precipitated with the addition of 3 volumes of ethanol in the presence of 0.04μg/μL glycogen. cDNA libraries were constructed using an Illumina-like protocol.
Results:
The quality of the cDNA libraries proved the quality and efficiency of the Precellys 24 when working with hypothalamic tissues. Precellys 24 homogenizer alone can be used at least for hypothalamic tissues. For other tough tissues requiring a longer time of homogenization, Cryolys cooling option is strongly recommended.
MiRNA expression in PVN profiles. Expression of the 20 most highly expressed miRNAs are shown for the five PVN profiles. Different miRNAs are shown in different colors.
