Hi all! We have been following a protocol for zebrafish larvae homogenization in PBS and measurement of glucose in the supernatant. Is there any way to isolate RNA from these samples? I have managed to isolate DNA from the pellet that is formed after centrifugation. Do you think that i would be able to isolate total RNA for qPCR from this pellet too? Has anyone ever done this before? Any help would be greatly appreciated!!
Try with TRIzol. I would recommend isolating RNA from both the supernatant and the pellet, as there may be different yields. In addition, by using the TRIZOL protocol you may be able to isolate DNA, RNA and proteins.
https://tools.thermofisher.com/content/sfs/manuals/trizol_ls_reagent.pdf
You can try it with trizol method..but u must get fragmented RNA.What is your purpose for RNA isolation???RT PCR??then that RNA may not work properly.
Because of the tendency of RNA to easily degrade, you would probably be better served by processing a portion of the sample for isolation of RNA and using second portion for glucose measurement.
If you were absolutely certain that glucose measurement reagents, which usually includes glucose oxidase, are free of RNAse, one might consider recovering RNA from used sample. However, even in such a case, you couldn't prevent the endogenous RNAse from degrading the RNA while glucose measurements were made. Addition of chaotropic agents, such as guanidinium isothiocyanate at high concentration will denature RNAse but it will also destroy the activity of glucose oxidase.
I would suggest splitting the sample in two portions, using minimum amount necessary for glucose measurement and focussing on optimizing recovery of RNA from the remaining portion of tissue sample.
Best,
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