Next Generation Sequencing Interest Group

Analysis of 14-3-3 proteins in the retina

Shivangi M. Inamdar, PhD
Assistant Research Specialist
Dept. of Biochemistry and Molecular Medicine, University of Iowa

14-3-3 proteins are small, ubiquitously expressed adaptor proteins that play an important role in several biological processes. Mammals express seven different isoforms that are expressed in a tissue specific manner. 14-3-3 proteins are expressed in the mammalian retina and have been implicated to play a role in photoreceptor function. However, which of the seven isoforms contributes to this function is not known. To study this, we used quantitative digital droplet PCR and immunohistochemistry and determined the differential expression levels and localization patterns of the 14-3-3 isoform proteins within the retina.

Using droplet digital PCR (ddPCR) to perform absolute quantification analyses of RNA transcripts and DNA targets from low input samples: Applications, workflow tips and tricks

William R. Kiffmeyer, PhD
Field Applications Specialist, Bio-Rad Laboratories Ltd.

While Real Time PCR remains the gold standard for the quantitative analysis of nucleic acids, droplet digital PCR offers quantitative measurements with greater sensitivity and precision. While the ddCq method of quantification REQUIRES validated efficiency calculations of the PCR reaction, ddPCR is less susceptible to inhibition and does not require validation by amplification efficiencies. We will compare and contrast the two technologies discussing ddPCR workflow and determining the absolute quantification of nucleic acids, without the need for standard curves, from precious samples. As an example of precious sample, we will touch on the quantification of targets in the Liquid Biopsy application. ddPCR allows for the sensitive and precise monitoring of rare targets in difficult matrices.