I'm doing a nuclear/cytoplasmic RNA extraction, i need an RNA that remain permanently in the nucleus and can be used as a control of the purification of the nuclear fraction. Thanks in advance for answers.
Dear Luigi,
There are methods to extract total RNA, mRNA, tRNA, but I don't know whether it is possible to isolate total or mRNA from cytoplasm and nucleus independently. It seems these methods would be difficult to make as RNA is a substance easy for rapid degradation. What might be possible is to make labelling/hybrysization of nucleic RNA and RealTime-RT-PCR measurement of hybrysized regions. But for that you need to know which RNA you are looking for, the sequence.
Greetings,
E.F.
You may try to use an intonic sequence of more or less abundant RNA . If you anchor your primers to the flanking parts of the exon, you may achieve your goal
I would imagine that the majority of the spliceosomal RNAs will stay in the nucleus (though a literature search suggests they're not necessarily exclusively resident there), but things like snoRNAs and so on should constitute a fairly decent pool for assessing nuclear/nucleolar purity.
Hi, are you separating the nuclear and cytoplasmic fractions before RNA extraction? If so, you could confirm the separation of the two fractions by detecting a nuclear protein vs a cytoplasmic protein. It would be an indirect way of confirming that the two fractions are pure, though I don't know if RNA would degrade in the purification process or "leak" from one fraction to the other.
Thank you all for the answers,
Following the suggestions I have identified the U2 snRNA (part of the spliceosome) which seems to be a good marker of the nuclear fraction, I'll try to use it!
If doesn't work then try to use the indirect method of protein (my protocol provides a first step of separation of the two fractions), although in this way it isn't possible to highlight a RNA degradation.
Luigi Cari I am interested in knowing if you find any potential marker for the cytoplasmic mRNA? I am considering H3 for the nuclei but not sure about the cytoplasm. Secondly is U2 snRNA works for you?
Looking forward to hearing from you.
Hi, have you already resolved this question for the nuclear RNA marker?
Dear Luigi Cari , I am also in the same condition. I have separated the nuclear and cytosolic fraction using Nuclear/Cytosol Fractionation Kit (Biovision Catalog #: K266), then using TRI reagent purified the total RNA and also generated cDNA using total RNA as template. Now I want to know which would be a positive and negative control for nuclear fraction as well as for cytoplasmic fraction as I have to perform PCR for my experiment to confirm the localization of my RNA of interest. Please share how you went ahead. It would be great help.
In my experiments, I was interested only to the nuclear fraction, and I reach good results using U2 snRNA; indeed, after nucleus/cytoplasm extraction, the nuclear fraction has a 20 times greater expression of this RNA in RT-qPCR (sybr green method).
Best!
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