Polysome Profiling

mRNAs are translated with different efficiencies which can change rapidly during adaptation to diverse environmental factors, including cellular stresses and microbial infections. Polysome profiling, the analysis of mRNAs associated with polysomes, has been widely used to investigate the post-transcriptional mechanisms (PTMs) that are involved in different biological processes. Creative Biolabs offers a comprehensive polysome profiling service for worldwide customers. This encompasses the use of a combination of RNA and protein analysis services and leverages the unsurpassed experience and expertise of our world-class team of molecular biologists and bioinformaticians.

Introduction

Gene expression is controlled by a series of mechanisms. Continuous findings regarding new regulatory mechanisms of gene expression are due to an increased understanding of RNA and more integrative analysis tools. Translation variants have already been shown as crucial determinants of mammalian gene expression but genome-wide expression profiling is not able to detect the fine adjustment provided by PTMs. To overcome this issue, the polysome profiling technique has been used to isolate and further independently analyze ribosome-free and polysome-bound RNAs. RNAs associated with many ribosomes, called polysomes, form large complexes of high molecular weight and can be easily segregated from ribosome-free RNAs through a sucrose gradient.

Polysome Profiling Applications

Different from ribosome profiling which captures positional information of ribosome footprints at the subcodon level, polysome profiling is more suitable for investigating alternative start codons or open reading frames. Polysome profiling analysis utilizes ultracentrifugation to isolate complexes of mRNAs in the process of translation and corresponding proteins based on density. Once each polysome fraction is collected, the translation activity of each mRNA could be analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray, or deep-sequencing.

Fig.1 Schematic representation of the sucrose gradient and representative polysome profile. (Pereira, 2018)

Services at Creative Biolabs

At Creative Biolabs, polysome profiling could also combine with microarray and RNA-sequencing for high-throughput screening. Major downstream analyses at Creative Biolabs are shown in Fig.2. Additionally, ribosome affinity purification followed by RNA analysis is an alternative that can be used for characterizing ribosome-bound RNAs. A successful polysome profiling should show clear 40S subunit, 60S subunit, 80S monosome, as well as several polysome peaks. The polysome peaks represent the RNAs bound to an increasing number of ribosomes. The robustness of polysome peaks is dependent on the proliferation state of the cell samples and varies between different cell lines. In general, at least three polysome peaks are observed for a good polysome profile.

Fig.2 Downstream processing of RNA and proteins obtained from polysome profiling. (Creative Biolabs)

Typical Procedure

1. Preparation of cell lysates
2. Preparation of sucrose gradients
3. Isolation of polysomes
4. RNA isolation and RT-qPCR MQ
5. Protein isolation and SDS-PAGE

Creative Biolabs combines bioinformatics and biology expertise to guarantee high-quality polysome profiling analysis services. If you are looking for polysome profiling services, please feel free to contact us for more information.

Reference

1. Pereira, I. T.; et al. Polysome profiling followed by RNA-seq of cardiac differentiation stages in hESCs. Scientific data. 2018, 5(1): 1-11.