Project protocol - Contents
- Workflow and sampling
- Equipment and supplies
- Reagents and solutions
- Procedure: microRNA preparation and quantification
- Definitions and calculations
- Data
Workflow and sampling
Mice euthanized; hippocampi dissected and snap frozen Total RNA preparation; microRNA isolation RT-PCR Equipment and supplies
- Dissection equipment
- 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City CA)
- Nanodrop ND-1000 (Thermo Fisher Scientific)
Reagents and solutions
- miRNA Isolation Kit (Ambion, Life Technologies, UK)
- TaqMan RT-PCR assays (Applied Biosystems, Foster City CA)
- Primers for selected microRNAs
Procedure: microRNA preparation and quantification
- Mice are euthanized by cervical dislocation.
- Bilateral hippocampi are dissected in their entirety from fresh brains within two minutes from the time of death.
- Connective tissue is trimmed off and hippocampi are immediately snap frozen on dry ice and stored at -80°C.
- Total RNA is isolated using a miRNA isolation kit.
- RNA concentration is determined using a Nanodrop ND-1000.
- RT-PCR reactions are performed in triplicate using 0.5 µL of 20x PCR Probe/Primer Mix, 1.5 µL of product from the RT reaction (diluted 1:10), 5 µL of 2x TaqMan Master Mix (no UNG), and 3 µL nuclease-free water.
- A sample minus reverse transcription buffer is used as a negative control.
- Reactions are run on a real time PCR system in 384-well format.
- RNU19, miR-9, and miR-99a are used as controls because their expression does not differ across strain and variability is low within strain.
- Relative expression is calculated using the comparative Ct relative expression method in Microsoft Excel; relative expression is normalized to the geometric mean of RNU19, miR-9, and miR-99a.
- microRNA relative expression for miR-15b, miR-31, miR-34c, miR-212, miR-201a