Abstract
Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and western blotting, genome-wide microarray analysis or qRT-PCR. Here, we describe a method for the isolation and quantification of high-quality polysome-bound mRNA complexes from small quantities of liquid-nitrogen-frozen solid tissue samples of rice shoots/roots. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level.
Materials and Reagents
Equipment
Software
Procedure
Representative data
We used a simple and highly reproducible method for the analysis of polysomes in rice (Oryza sativa). All the experiments we made, displayed a very similar profiles, however, some differences can be observed in the form of the profiles when the plants are stressed and also in the older plants. In the literature, analysis of polysome profiles issued from different plant species show a variability between species and plant organs as shown in Figure 2 below. Figure 2. Analysis of polysome profiles from different plant species Example 1 (Figure 2a): Polysomal extrcation from Medicago truncatula roots. Ribosomes were pelleted by ultracentrifugation of whole extracts (S-16) of transgenic roots through a 1.7-m sucrose cushion to obtain the post-ribosomal supernatant (S-170) and the ribosome pellet (P-170). P-170 was fractionated by ultracentrifugation through a 20–60% (w/v) sucrose density gradient and the absorbance at 254 nm was recorded. Positions of monosomes (80S) and large polysomes (LP, ≥5 ribosomes) are indicated. [from Reynoso et al. (2012)] Example 2 (Figure 2b-c): Ribosomes purified from Arabidopsis tahliana leaf tissues by conventional ultracentrifugation (P-170) (a) or by immunopurification (eluate) (b) were fractionated in 20% to 60% (w/v) sucrose density gradients and the UV absorbance (254 nm) profile was recorded. The positions of the 60S ribosomal subunit, 80S mono-somes, and polysomes are indicated. [from Zanetti et al. (2005)] Example 3 (Figure 2d): Cadmium-induced alteration of the polysomes profile using Arabidopsis thaliana suspension cells . The polysome profile of control and cadmium-treated cells treated with 200 μM cadmium for 4 or 12 h, are represented. [from Sormani et al. (2011)] Example 4 (Figure 2e): Sucrose density profiles of ribosomes and polysomes from Pisum sativum. Ribosomes recovered after 6 h. centrifugation through 1 M-sucrose cushion. E254=absorbance at 254 nm. [from Leaver and Dyer (1974)] Example 5 (Figure 2f-g): Typical polysome profiles obtained from tobacco leaf tissues and protoplasts, respectively, are shown in (a). The effect of detachment on the polysome populations of tobacco leaves is schown un (b). Sucrose density gradient profiles of polysome preparations obtained from attached leaves (left), leaves detached and floated on water for 4 h (middle) and leaves detached and floated on 0.6 M mannitol for 4 h (right). [from Ruzicska et al. (1979)] Example 6 (Figure 2h): Polysome profiles generated from roots of young WT rice plants grown in Pi-sufficient media (15 d-old). [from Jabnoune (unpublished)] Figure 3. Fractionation of polysome gradients Figure 4. A representative example of data obtained Note: We used a simple and highly reproducible method for the analysis of polysomes in rice (Oryza sativa). All the experiments we made, displayed a very similar profiles, however, some differences can be observed in the form of the profiles when the plants are stressed and also in the older plants. In the literature, analysis of polysome profiles issued from different plant species show a variability between species and plant organs (see examples in Figure 2).
Notes
To isolate the RNA form fractions from sucrose density centrifugation for polysome analysis, it’s important to keep in mind that TRizol purification could be the best option in case of large scale extraction (Rice produce more biological materiel than Arabidospis). In case of small samples (particularly Arabidopsis roots samples) the use of RNeasy kit purification of RNA will be more cost-effective but sometimes the quality of the RNA would be not of high quality for subsequent qPCR analysis. It’s important to centrifuge the Trizol extract at 12,000 x g for 15 min to separate the phases. We have to sacrifice a layer of the aqueous phase (containing RNA) at the interphase, so that we will carry over proteins and phenol to the RNA.
Recipes
Acknowledgments
This work was funded by the Swiss National Foundation grant (31003A-12293 and 31003A-138339) and the Sino-Swiss Science and Technology Cooperation Program (IZLCZ3 123946 to YP and 2009DFA32040 to QS).
References
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