This protocol is significant because it is, to our knowledge, the first one to describe a new centrifuge based metal for kinetic polysome purification from soybean symbiotic nodules. The big advantage of this technique is that in combination with RNA-Seq, allows the study of the relation and organized in a complex tissue such as the symbiotic nodule. To begin, weigh approximately 0.2 grams of intact nodules in a weighing dish, and transfer them to a pre-cooled two milliliter tube.
Then add 1.2 milliliters of polysome extraction buffer, let the sample thaw for two minutes, and homogenize with a tissue grinder until complete disruption and homogenization of the nodules. Next, incubate the samples on ice with gentle agitation for 10 minutes or until all samples have been processed and centrifuge at 16, 000 times G for 15 minutes at four degrees Celsius to pellet the debris. Then, recover the supernatant, and repeat the centrifuge step.
After carefully recovering the clarified cytosolic extract, transfer a 200 microliter aliquot to a clean 1.5 milliliter micro centrifuge tube for total isolation. Then, pour 4.5 milliliters of the 33.5%sucrose layer into the centrifuge tubes, and add 4.5 milliliters of the 12%layer carefully and slowly with a P-1000 micropipette. Next, put the tubes on ice until the addition of the clarified cytosolic extract.
Load one milliliter of the clarified cytosolic extract on top of the sucrose cushion by carefully pipetting onto the side wall of the tube. Then, transfer the ultra centrifuge tubes to pre-cooled buckets, and centrifuge at 217, 874 G for two hours at four degrees Celsius. After discarding the remaining cytosolic extract and sucrose cushion, re-suspend the polysomal pellet with 200 microliters of pre-cooled re-suspension buffer.
Next, incubate for 30 minutes on ice, and then transfer the polysomal re-suspension to 1.5 milliliter pre-cooled tubes to proceed with the RNA extraction. After homogenizing the samples with 750 microliters of the RNA isolation reagent incubate for five minutes at room temperature. Then, add 200 microliters of cold chloroform, and shake the tubes vigorously for 15 seconds.
Next, incubate at room temperature for 10 minutes. Centrifuge at 12, 000 times G for 15 minutes at four degrees Celsius for phase separation, and transfer 500 microliters of the upper aqueous phase to a clean tube without disturbing the pink organic phase. Then, add 375 microliters of cold isopropanol and 0.5 microliters of RNAs free glycogen.
Afterward, mix thoroughly by pipetting up and down. Incubate the mix for 10 minutes at four degrees Celsius, and centrifuge at 12, 000 times G for 15 minutes. Then, discard the supernatant, and wash the RNA precipitate with one milliliter of cold, 75%ethanol.
Mix by brief vortexing. Next, centrifuge at 7, 500 G for five minutes at four degrees Celsius. Discard the supernatant, and air dry the RNA pellet.
Then, dissolve the pellet in 50 microliters of RNAs free water, and incubate at 65 degrees Celsius for five minutes. Assess the RNA concentration and integrity with highly sensitive capillary electrophoresis and, or electrophoresis on a 2%RNAs free agarose gel. After estimating the sample volume, add 0.1 volumes of three molar sodium acetate, three volumes of cold ethanol, and 0.5 microliters of RNAs, free glycogen.
Then, mix them thoroughly. Capillary electrophoresis was performed for several total and polysomal associated RNA sample fractions, which demonstrated sharp ribosomal RNA bands, without any smeared appearance. However, this is not reflected in the RIN values, as they range between 5.9 and 7.5, which corresponds to non-intact samples.
The bioanalyzer failed to identify the 18S and 25S peaks as seen in the electropherograms, both for total and polysome associated RNA samples. There was no visual sign of sample degradation. However, a sample was analyzed to visualize the result of almost entirely degraded sample, for comparison.
The most important thing is to work in polysome and RNA preserving conditions during the whole protocol. After sequencing the total and par RNA fractions standard patterns for the gene expression analyzed can be used to identify differential express genes at the transcription and the translation level.
