Light sensitivity

The first major obstacle to the study of ocular tissues is their light-sensitivity. The manner in which ocular tissues are collected may alter the expression of the transcripts of interest. This scenario is not unique to the eye. As changes in gene expression are dynamic, the methods used to dissect tissues or dissociate cell types may consequently affect the expression of either target or internal control genes (Dougherty and Geschwind, 2005). This technical consideration is rarely controlled for, and may result in potentially misleading data. These problems are compounded when temporal changes in gene

DR IR

DR IR

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Ol o

Blue cone Rod

Green cone

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10.0000

IS 0.0100

Ol o

0.0010

0.0001

DR IR

DR IR

10.0000

IS 0.0100

Ol o

0.0010

0.0001

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UV cone Rod

Green cone

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Figure 6.1

Spectral sensitivity profiles for human A. and mouse B. visual pigments. Data plotted on logarithmic scale. Arrows indicate dim red stimulus, at 600 nm (DR) and longer-wavelength infra-red stimulus at 700 nm (IR), illustrating the increased long-wavelength sensitivity of the human visual system conferred by the presence of the red cone class. Visual pigment profiles based upon Govardoskii template (Govardovskii et al., 2000). ^max values taken from Dartnall et al. (1983), Jacobs et al. (1991), Bridges (1959) and Sun et al. (1997).

expression are of interest, as not only tissues must be collected without light exposure, but they must be collected rapidly to prevent changes in gene expression or RNA degradation (see 'RNA quality' below).

Visual scientists have traditionally collected tissues under dim red light (>600 nm) to minimize the effects of photopigment activation. For many studies, this works extremely well, although it should be noted that the success of this approach is dependent upon the species and its innate photoreceptor complement in comparison to human spectral sensitivity. The visual sensitivity functions of photopigments can extend well into the long-wavelength region of the spectrum and use of even dim red light may result in a significant absorption by the LWS cones of some species. Figure 6.1 illustrates the photopigment complements of humans (A) and mice (B), illustrating the difference in long wave sensitivity between these species.

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