28S:18S rRNA ratio

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Nite
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28S:18S rRNA ratio

Post by Nite » Thu Jul 27, 2006 10:01 am

The ratio of 28S:18S rRNA in gel electrophoresis of sample containing total RNA is theoretically 2:1 when the sample rRNA integrity is high (ie. the rRNA is intact). What are the reasons behind this observation?

I have googled the thing but I din find any explanation.


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Post by MrMistery » Thu Jul 27, 2006 6:45 pm

What do you mean? If the rRNA molecule breaks, more bands will show up on your gel. I don't understand your question..
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Post by Navin » Fri Jul 28, 2006 7:05 am

i think nite means "why is there twice the amount of 28s as compared to 18s (after running the gel)".
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Post by Nite » Fri Jul 28, 2006 4:11 pm

yes what navin said is almost correct..

wad i mean is..

after we extract out total RNA from a cell sample.. then we PCR the sample.. then we send the sample for gel electrophoresis. Two (or maybe three) bands will be observed. One band will be the 28S rRNA band, the other is 18S rRNA ( and the third one maybe 5S or 5.8S).

On the gel, the putative observation when the total RNA extract is of 'good' quality is that the 28S band will always have twice the intensity of 18S. Why is this so?
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Post by canalon » Fri Jul 28, 2006 5:55 pm

Same number of molecules, but twice the size, hence twice more EtBr binding site.
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Post by Nite » Sat Jul 29, 2006 2:08 pm

hm.. ?? I do not think that the size of the molecule has much effect on the band intensity in this case? Becoz if the size had substantial influence, the marker DNA that were added would have shown a gradient of band intensity. But that does not happen rite..
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Post by canalon » Sat Jul 29, 2006 2:20 pm

Well since in the old time we used to dose DNA by intensity of the band of the gel, I know that the size of the molecule has a huge effect on the intensity. In fact it is still a quick and dirty way to compare the efficiency of some reactions for example, when i can't be bothered to go use the spectrophotometer or the fluorimeter.
In fact EtBr insert itself every 10 bases (about) at saturation. And fluorescence is directly proportional to the number of EtBr molecules. So fluorescence depends on the number of molecules AND their individual size. This is a linear relation ship so a same number of molecules, twice as big will have a double intensity.

And if you give a close look at your marker information sheet you will see that the concentrations are constant all along the size.
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