What does nuclear total RNA look like?

[pmiguel]

Has anyone run BioAnalyzer chips on total RNA isolated from nuclei? I am seeing a broad peak at around 5000 bases and nothing that looks like an 18S peak and am not sure what to make of it. (This is from drosophila nuclei, so for cytoplasmic rRNA, most or all of the 28S is cleaved into pieces the same size as the 18S...)

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Phillip

[Genohub]

What approach did you take to isolate your total RNA? Could the 5kb peak be genomic DNA or did you do a DNase digest? Have you ruled out mtDNA/RNA?

- Genohub

Has anyone run BioAnalyzer chips on total RNA isolated from nuclei? I am seeing a broad peak at around 5000 bases and nothing that looks like an 18S peak and am not sure what to make of it. (This is from drosophila nuclei, so for cytoplasmic rRNA, most or all of the 28S is cleaved into pieces the same size as the 18S...)

--
Phillip

[pmiguel]

What approach did you take to isolate your total RNA? Could the 5kb peak be genomic DNA or did you do a DNase digest? Have you ruled out mtDNA/RNA?

- Genohub

Not our prep. The customer treated the sample with DNAse, so hopefully no genomic DNA left after that. Unless the DNAse failed to work.

Agilent mentioned that residual RNA Zap could cause this sort of peak on a pico chip.

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Phillip

[Genohub]

You can use this primer set (180 bp product) to quickly assess whether its mtDNA:

Forward -AATCCAAGCCTACGTTTTCACA
Reverse -AGTATGAGGAGCGTTATGGAGT

I've never heard about residual RNA Zap causing that to the trace, interesting. Can you upload the bioanalyzer image?

- Genohub

[pmiguel]

You can use this primer set (180 bp product) to quickly assess whether its mtDNA:

Forward -AATCCAAGCCTACGTTTTCACA
Reverse -AGTATGAGGAGCGTTATGGAGT

I've never heard about residual RNA Zap causing that to the trace, interesting. Can you upload the bioanalyzer image?

- Genohub

Sure:



The pico chip says the sample is 713 pg/ul. Sample was diluted 1:10 prior to loading. So about 7 ng/ul. Probably should just run it on a nano chip. They are less prone to artifacts.

[Genohub]

I agree, they certainly don't look like typical discrete 18S or 28S peaks. It doesn't look like electrode cartridge contamination or bubbling either. Typically peaks like this are genomic DNA. I would take a small amount and re-digest with DNAse to verify.

Yes, the nano chip tends to have fewer artifacts in my experience too.

- Genohub

[firsal]

What does nuclear total RNA look like?

Should I see the 18s and 28s peak when I running RNA BioAnalyser chips for Nuclei RNA? I don't see any peak in my run. Is that mean my nuclei RNA is degraded?

[pmiguel]

Should I see the 18s and 28s peak when I running RNA BioAnalyser chips for Nuclei RNA? I don't see any peak in my run. Is that mean my nuclei RNA is degraded?

Either degraded, or at a concentration below the limits of detection of the chip you ran. Did you run a nano or pico chip?

--
Phillip

[firsal]

What does nuclear total RNA look like?

Either degraded, or at a concentration below the limits of detection of the chip you ran. Did you run a nano or pico chip?

--
Phillip

I ran it on nano chip. This is RNA isolated from 10,000 nuclei.

[pmiguel]

I ran it on nano chip. This is RNA isolated from 10,000 nuclei.

Did you see anything in the trace, or was it completely flat?

--
Phillip

[firsal]

What does nuclear total RNA look like?

Did you see anything in the trace, or was it completely flat?

--
Phillip



I enclosed the image of my result in attachment. It shows a wide peak of RNA from 100-2500 nucleotide.

[pmiguel]

If that is total RNA then it is degraded.
Some protocols would still work for RNAseq. Like NuGEN or ribo-depletion.
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Phillip

[firsal]

If that is total RNA then it is degraded.
Some protocols would still work for RNAseq. Like NuGEN or ribo-depletion.
--
Phillip

Thanks for your comments. I'll try them. Could be that I've isolated nuclei from frozen cells in -80?

[pmiguel]

Thanks for your comments. I'll try them. Could be that I've isolated nuclei from frozen cells in -80?

I'm not following what you are asking here.

--
Phillip

[firsal]

I'm not following what you are asking here.

--
Phillip

I'm sorry if I was not clear. I have frozen cells isolated from adipose tissue that I store them at -80 freezer. I freeze cells without any buffer. I do nuclei isolation from 5 ml of frozen cells by adding same volume of lysis buffer.

I have used this isolated nuclei to get genomic DNA and it worked nicely but I'm not sure if this precedure is good to get good quality of RNA.