The trade-off between using discrete tissue masses containing multiple cell types or attempting to reduce cellular heterogeneity and possibly affecting
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Analysis of RNA quality using Agilent Bioanalyser with RNANano chip. A. 500 ng of total RNA was loaded per well, producing two distinct peaks for the 18S and 28S ribosomal RNA subspecies. B. In degraded samples, RNA fragmentation produces a shift to smaller molecular weight products. Data courtesy of Helen Banks.
gene expression or RNA quality presents a major problem with no simple solution. Without extensive validation to establish the suitability of dissociation/dissection techniques in a variety of experimental scenarios, the answer remains unclear. Approaches such as fluorescent-assisted cell sorting (FACS) and laser capture microdissection (LCM) offer potential solutions to these problems, but validation for specific research applications would still be required. The best advice would be to ensure that whatever approach is used, when comparisons are made between tissues both should be isolated in a similar manner.
Preliminary analysis of c-fos induction within the murine retina using whole eye or just retinae suggests that both approaches can detect the increase in expression (Semo et al., 2003). However, the amplitude of the change is lower in the whole eye (1.6-fold increase relative to sham) compared with just retinae (6.8-fold increase) as would be predicted due to a smaller contribution to the total RNA pool (Dougherty and Geschwind, 2005).
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