rRNA (28s and 18s) appear smaller on gel

[Rindfleisch]

hi,
today I´ve run a normal non-denaturating 1% agarose gel with RNA (~180ng) isolated from jurkat cells for quality and integrity assessment.
normally the 28srRNA band should have twice the intensity then the 18srRNA band, but this not the case for me....its even the other way round. Furthermore the two RNA-molecules appear to be too small of size. 28sRNA is approximately 1,5 kb big and 18 srRNA around 500bp. This is usually a sign of RNA-degradation, right? I really dont know why this should be the case because i worked very cleanly and thouroughfully. i even denaturated my RNA at 70°C for 1 minute before applying the RNA onto the gel.

For me there are two explanations:
1) i eluted in nuclease-free water (ambion)
2) jurkat cells are tumor cells and those have in general degraded rRNA

help and thoughts are appreciated.

[relaxin]

What do you use for RNA size markers? If you use DNA size markers, the size of the rRNA may not be correct.

[Rindfleisch]

i used 100bp and a 1kb ladder, which i normally for the determination of the size of dna-molecules.
didnt know there are rna-ladders..

do you think, for quality assessment its sufficient to see two sharp bands without smearings?

[relaxin]

Yes, there are RNA size markers. They are available from companies such as Ambion.

If you see two sharp bands of rRNA, your RNA quality should be good. You may also see a smear of mRNA of different sizes.

[morkfromork]

hi,
today I´ve run a normal non-denaturating 1% agarose gel with RNA (~180ng) isolated from jurkat cells for quality and integrity assessment.
normally the 28srRNA band should have twice the intensity then the 18srRNA band, but this not the case for me....its even the other way round. Furthermore the two RNA-molecules appear to be too small of size. 28sRNA is approximately 1,5 kb big and 18 srRNA around 500bp. This is usually a sign of RNA-degradation, right? I really dont know why this should be the case because i worked very cleanly and thouroughfully. i even denaturated my RNA at 70°C for 1 minute before applying the RNA onto the gel.

For me there are two explanations:
1) i eluted in nuclease-free water (ambion)
2) jurkat cells are tumor cells and those have in general degraded rRNA

help and thoughts are appreciated.

Hi there,
I'm a bit confused about your protocol, you say you ran a non-denaturing gel, but you heated your RNA samples to denature before running them. Usually if I want to check RNA quality I just run about 1ug on a regular TAE-1% Agarose gel , no heating required (pre-treat the gel tank with NaOH and then rinse very extensively with Milli-Q H2O to make it clean). For mouse RNA 28S runs at around 2kb (dsDNA ladder) and 18S runs around 1kb.

Degraded RNA would look like a smear, not necessarily a size shift. Because the rRNAs have secondary structure, you can't expect them to run at their predicted size. You would need to run a MOPS-Formaldehyde gel with RNA markers.

If your rRNA looks like distinct bands your RNA is most likely OK.

[Rindfleisch]

thanks a lot all of you.
i have two sharp bands...but next time i try with a rna-ladder.

[.anka]

Hi,

I ısolated total RNA from FFPE colon tissue with FFPE RNeasy Kit and run my sample 1% native agarose gel but I saw just one sharp band. Today run it again but this time I incubated sample at 70 C and then I took it on ıce before loading, however I saw again one band.

So what should I to see both of 28S RNA and 18S RNA?

Thanks

[nootropic]

In the course of obtaining tRNA for cell-free protein synthesis, I performed simple phenol-solo extraction on cells of E. coli and ran a non-denaturing 1% agarose gel, resulting in the picture shown below.


Lane 1: 1 kb DNA Ladder
Lane 2: Sample, undiluted (1,63 µg/µL)
Lane 3: Sample, 1:10
Lane 4: Sample, 1:100
Lane 5: 100 bp DNA Ladder

In view of the bands on their own, I would interpret the two bands in the middle to represent the 23S and 16S ribosomal subunits and the lowest band, which in contrast to the others also appears as smear, to represent the tRNA population. But in view of the ladders, there appear significant differences between the sizes that are indicated on the gel and those that were excepted. While I can imagine that the difference of about 150 - 200 bp for the tRNA-bands can be explained by their secondary structure-related mobility shift, I don't know how to explain the position of what are supposed to be the 23S and 16S bands. The 23S is about 2900 nt long but appears at 1500 bp and the 16S is 1500 nt long but appears at about 1000 bp. Is this the reason of using a DNA marker instead of one for RNA? is it because of secondary structure influence? Or am I even false with my interpretation of the bands after all?
Further purification of tRNA is intended to be done by sucrose gradient centrifugation

[mchlbrmn]

Yes. As you suggest you cannot compare a double stranded DNA size ladder to single stranded RNA, which in addition to being 1/2 the mass due to having only one strand also folds into various conformations. RNA size ladders can be purchased.
Perhaps the band at the top is undissolved RNA?
I haven't run RNA on gels for many years, but Mork above in the thread says that on a TAE gel 28S runs about like 2 kb DNA, and 18S as 1kb. Adjusting for differences in the size from mammalian to bacteria, I think this means your sizes may be roughly correct.
I've never worked with tRNA, but I would guess that a higher percentage would get a sharper tRNA band, and might have a better yield by losing less to diffusion (unless the extra agarose inhibits the purification?).

[simbu]

hi friends,

when i ran MOPS -formaldehyde agarose gel, my 28s and 18s rRNA bands always seems to be similar. can you give some suggestion why it so? on which step i have to improve to get it right? i am looking for your kind reply.

thanks
simbu

[mchlbrmn]

I can't understand the question. Do you mean that you only see one band?