I am looking to detect my transcript of interest in the brain ring gland complex in polysome fractions and I am wondering what is the minimum amount of dissections I will need to do. Thanks all!
The minimum amount will depend a lot on what do you want to do with your polysome-bound RNA. If you want to check it with RT-qPCR you need very little, if you want to do northern blot you need a lot...... Which means that your question does not have an easy an straight-forward answer.
It also depends whether you want to do a polysome gradient (10-40% sucrose) or you can work with a step gradient (30% sucrose) where all RNA bound to ribosomes will enter the gradient, but it can be carried out in smaller volumes, and since you collect it on a single fraction you would need less RNA.
I believe that you have to give more specifics. We have done polysome gradients with mouse brain, mammary gland, etc and we used on the order of milligrams on the best cases. To detect some RNAs you might need more
Thanks for the reply,
I am looking to do qPCR and determine whether my transcript as polysome-bound RNA is present or absent at certain time points in development. I am new to this type of procedure, but the second option of the step gradient sounds like it may suit my needs.
thanks again!
For the one step gradient you can have a look at a Faseb Journal manuscript of ours (Garcia-Sanz et al., 1998). For the use of tissue RNA for gradients delPrete et al., (RNA) and the description of the polysome gradients from the Methods on Immunology Manual (chapter 7.8 -I believe-) you will find all of them in Linkedin
If you have any further questions or queries, do not hesitate to send a message
best
Jose A.
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