Hello, I have some questions for you. 1. After preparing the...

Thank you for your questions.
1. We do not typically measure the RNA purity after extraction from the collected polysome fractions, so we cannot speak to the expected A260/280 values. Instead, we use the same uniform volumes of each fraction for the analysis. We prepare RNA from the same volume of each fraction, then prepare cDNA from this RNA using the same volumes, all side by side without measuring RNA concentrations. After RT-qPCR for each fraction, we then calculate the measured mRNA in each fraction as the % of that mRNA in the entire gradient. You may want to try RT-qPCR with primers for a housekeeping mRNA (GAPDH or ACTB) to determine: a) if the % mRNA distribution curves look similar to your specific mRNA, this would suggest an experimental problem with the gradient or centrifugation, or
b) if the % mRNA distribution curves for the housekeeping mRNA look more as expected, then perhaps the issue resides with the detection of your specific mRNA.

2. It is challenging to diagnose whether the gradient or centrifugation was problematic without seeing the global polysome profile with ribosome peaks. The PCR results from your specific RNA, if these represent the % mRNA distribution across the gradient, suggest that the polysomes might be compressed at the bottom of your gradient – either improper sucrose compositions of the gradient and/or centrifugation for too long may cause this. Alternatively, the bottom portion of your sucrose gradient was not actually collected, so your fractions did not contain the entire gradient and all of the polysomes. You might collect an extra fraction or two next time, just to be sure that you are analyzing the entire gradient - there should be little or no RNA in the last fraction or two.

We are sorry that we cannot more specifically identify the answers to your questions. Best of luck with your studies!