Email updates

Keep up to date with the latest news and content from RB&E and BioMed Central.

Open Access Highly Accessed Methodology

Quantitative analysis of mRNA translation in mammalian spermatogenic cells with sucrose and Nycodenz gradients

Kenneth C Kleene1*, Jana Bagarova12, Sabrina K Hawthorne1 and Leah M Catado13

Author Affiliations

1 Department of Biology, University of Massachusetts, Boston, MA 02125-3393, USA

2 Cardiovascular Research Center, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114, USA

3 Upper School Science Department, Buckingham, Browne and Nichols School, 80 Gerry's Landing Road, Cambridge, MA 02138, USA

For all author emails, please log on.

Reproductive Biology and Endocrinology 2010, 8:155  doi:10.1186/1477-7827-8-155

Published: 25 December 2010

Abstract

Background

Developmental and global regulation of mRNA translation plays a major role in regulating gene expression in mammalian spermatogenic cells. Sucrose gradients are widely used to analyze mRNA translation. Unfortunately, the information from sucrose gradient experiments is often compromised by the absence of quantification and absorbance tracings, and confusion about the basic properties of sucrose gradients.

Methods

The Additional Materials contain detailed protocols for the preparation and analysis of sucrose and Nycodenz gradients, obtaining absorbance tracings of sucrose gradients, aligning tracings and fractions, and extraction of equal proportions of RNA from all fractions.

Results

The techniques described here have produced consistent measurements despite changes in personnel and minor variations in RNA extraction, gradient analysis, and mRNA quantification, and describes for the first time potential problems in using gradients to analyze mRNA translation in purified spermatogenic cells.

Conclusions

Accurate quantification of the proportion of polysomal mRNA is useful in comparing translational activity at different developmental stages, different mRNAs, different techniques and different laboratories. The techniques described here are sufficiently accurate to elucidate the contributions of multiple regulatory elements of variable strength in regulating translation of the sperm mitochondria associated cysteine-rich protein (Smcp) mRNA in transgenic mice.