Tissue collection

For light-sensitive tissues, such as the eye, procedures should ideally be conducted under infra-red light (>700 nm). Infra-red optical devices are available from a wide range of suppliers, although complicated dissection is extremely difficult using such devices, and the exact wavelength of emission may vary. Lacking such facilities, dim red light may be used if a suitable cutoff filter is used to prevent stimulation of the photopigments present (see Figure 6.1). Based on the known long-wavelength sensitivity of human and mouse visual pigments, use of a light source with a cut-off of 600 nm would result in absorption of around 60% by the human red cone (certainly enough to see by), but only around 5% by the mouse green cone, or around a ten-fold greater sensitivity. Moving up to over 700 nm would result in less than 1% absorption by the human red cone (detectable at very high intensities), but over a hundred-fold less absorption by the mouse visual pigments.

Snap-freezing tissues is ideal for RNA extraction, although all dissections must be conducted before doing so as freeze-thawing can have dramatic effects on RNA quality (Sambrook and Russell, 2001). One solution to this problem is the use of an RNA preservation medium, such as RNAlater™ (Ambion). With ocular tissues, the eye can be enucleated under infra-red light, the eye pierced to facilitate access to the retina, and then placed into RNAlater™ in a light-tight container. The tissue can then be stored overnight at 4°C before the retina is dissected out in light.

To validate this approach, we investigated expression of the immediate-early gene and marker of neuronal activity c-fos in response to a 15 min light pulse. C-fos expression has been shown to rapidly increase in response to such a stimulus, corresponding to depolarization of retinal ganglion cells (Nir and Agarwal, 1993; Yoshida et al., 1993). Dissection of tissues stored in RNAlater™ followed by qPCR using SYBR® Green I results in a clear induction of c-fos, peaking at 30 min, as demonstrated previously (Figure 6.5).

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