Torrey Pines Institute for Molecular Studies

About: Torrey Pines Institute for Molecular Studies is a nonprofit organization based out in San Diego, California, United States. It is known for research contribution in the topics: Antigen & T cell. The organization has 2323 authors who have published 2217 publications receiving 112618 citations.

Protein instability following transport or storage on dry ice

Abstract: properties of transcription2,6 (Supplementary Note 4). To validate these methods, we first used simulations (Supplementary Note 5). For transcription sites smaller than the optical resolution, all methods yielded accurate estimates. However, for larger transcription sites, the simple methods led to gross underestimates, whereas FISH-quant gave accurate results (Fig. 1e). For elongated transcription sites, only the PSF superposition approach worked reliably. For experimental validation, we used an artificial reporter with transcription sites frequently exceeding the resolution. An RNase protection assay provided a rough, but independent, estimate of the ratio of mature versus nascent mRNA. The assay yielded ratios in the same range as the FISH-based quantifications, confirming their general validity (Supplementary Note 6 and Supplementary Methods). For a more accurate assessment of simple methods and FISH-quant, we then compared the nascent transcript counts. Much as for the large simulated transcription sites, the simple methods led to underestimated counts (Fig. 1f). Thus, FISH-quant accurately quantified nascent mRNA even when simple approaches did not. Finally, we used FISH-quant to analyze b-actin mRNA after serum induction and measured more than twice the amount of nascent mRNA than we did with simple methods, which illustrates the importance of accurate quantification even for endogenous genes (Supplementary Note 6 and Supplementary Methods). FISH-quant could also be applied to other structures with a dense accumulation of mRNA, such as processing (P)-bodies or stress granules. FISH-quant is controlled via graphical user interfaces in Matlab and requires no computational expertise. A batch mode allows users to automatically process multiple images. FISH-quant is available at http://code.google.com/p/fish-quant/ with a detailed manual and test data.