Schalkwyk2 project protocol

Hippocampal microRNA expression in 24 BXD recombinant inbred strains of mice   (2012)

Schalkwyk LC, Chesler EJ, Fernandes C, Liu L, Parsons MJ, Paya-Cano JL
With: Grimm C, Nietfeld W, Lehrach H


Project protocol - Contents

Workflow and sampling

Procedure performed
Data collected
Mice euthanized; hippocampi dissected and snap frozen
Total RNA preparation; microRNA isolation
microRNA relative expression

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

    1. Mice are euthanized by cervical dislocation.
    2. Bilateral hippocampi are dissected in their entirety from fresh brains within two minutes from the time of death.
    3. Connective tissue is trimmed off and hippocampi are immediately snap frozen on dry ice and stored at -80°C.
    4. Total RNA is isolated using a miRNA isolation kit.
    5. RNA concentration is determined using a Nanodrop ND-1000.
    6. 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.
    7. A sample minus reverse transcription buffer is used as a negative control.
    8. Reactions are run on a real time PCR system in 384-well format.
    9. RNU19, miR-9, and miR-99a are used as controls because their expression does not differ across strain and variability is low within strain.
    10. 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.

Definitions and calculations

  • miR = miRNA = microRNA

Data collected by investigator

  • microRNA relative expression for miR-15b, miR-31, miR-34c, miR-212, miR-201a