7 replies to this topic

[jamestoon1]

Hi.

I isolated RNA from human tissue samples using RNeasy.
I electrophoresed the RNA obtained and this is what I got:

P.S. 100bp DNA ladder was used instead of RNA ladder because we don't have RNA ladder in our lab.

From the gel image, two clear bands can be seen (supposed to be 18S and 28S rRNA).
But there are also some smaller bands down there.
Does this indicate RNA degradation or something else?

P.S. The tissue samples were preserved in RNAlater for 1 month before RNA isolation.

P.S. We don't have Bioanalyzer in the lab, so I cannot analyse it in the instrument.

P.S. I did not check the purity and concentration of the RNA because the only spectrophotometer in our lab has been send for repair.

Edited by jamestoon1, 19 January 2013 - 09:46 AM.

[bob1]

Degradation presents as a smear.  Bands are other rRNA bands

[jamestoon1]

bob1, on 19 January 2013 - 07:44 PM, said:

Degradation presents as a smear.  Bands are other rRNA bands

Thanks for the reply.
But I have seen other people's RNA gel image, and most of them got something like this (only 2 clear bands without those smaller bands):

So why are those other rRNA bands present only in my samples?
Does the presence of those other rRNA bands indicate something good or something bad?

Edited by jamestoon1, 19 January 2013 - 10:12 PM.

[bob1]

Usually good - it indicates that your RNA is intact and high abundance (though this is relative to the amount loaded on the gel).

[jamestoon1]

Thanks for the reply.

[almost a doctor]

In our lab, we have observed that with kits that use spin columns is normal to loose the smaller RNAs (they are not retained in the column), while those are still there with Trizol.  

Back to your picture, to me it looks like you do have some degradation, also may have some DNA contamination (top of the gel, still in the well).  It also looks like the gel is maybe overloaded and what I think is degradation (smeary bit between distinct bands) might just be due to too much RNA.

[jamestoon1]

almost a doctor, on 21 January 2013 - 07:44 AM, said:

In our lab, we have observed that with kits that use spin columns is normal to loose the smaller RNAs (they are not retained in the column), while those are still there with Trizol.  

Back to your picture, to me it looks like you do have some degradation, also may have some DNA contamination (top of the gel, still in the well).  It also looks like the gel is maybe overloaded and what I think is degradation (smeary bit between distinct bands) might just be due to too much RNA.

But I have read somewhere that if we isolate RNA with Trizol, the RNA integrity tends to be lower than isolating with RNeasy kit.
So if I need both small RNA and high integrity RNA for my work, based on your experience, would you suggest me to use Trizol or RNeasy?

I loaded 4ul RNA into the gel, and I only came to know later that for RNA, 2ul is sufficient, so most probably the smear is due to overloading of RNA.
But I'll run gel electrophoresis again after this.
DNA contamination is not unexpected because I did not use DNase this time (because this is only a trial experiment of mine).
I have failed several times in the past for my RNA isolation, and this is the first time I succeeded.
It turned out that my previous failed attempts was due to insufficient grinding of the tissues.

[Wickerl3732]

almost a doctor, on 21 January 2013 - 07:44 AM, said:

In our lab, we have observed that with kits that use spin columns is normal to loose the smaller RNAs (they are not retained in the column), while those are still there with Trizol.  

Back to your picture, to me it looks like you do have some degradation, also may have some DNA contamination (top of the gel, still in the well).  It also looks like the gel is maybe overloaded and what I think is degradation (smeary bit between distinct bands) might just be due to too much RNA.

I would agree with that, that you gave (I) DNA contamination and (ii) overloaded the gel. Think you have to load less, and if you still see smear below the 28 and 18S, it might be degraded to q certain extend. But the sharp bands would not argue for degradation.