Viewing 6 reply threads
  • Author
    Posts
    • #5334
      Nite
      Participant

      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.

      cheers

      nite

    • #52249
      MrMistery
      Participant

      What do you mean? If the rRNA molecule breaks, more bands will show up on your gel. I don’t understand your question..

    • #52282
      Navin
      Participant

      i think nite means "why is there twice the amount of 28s as compared to 18s (after running the gel)".

    • #52299
      Nite
      Participant

      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?

    • #52302
      canalon
      Participant

      Same number of molecules, but twice the size, hence twice more EtBr binding site.

    • #52351
      Nite
      Participant

      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..

    • #52353
      canalon
      Participant

      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.

Viewing 6 reply threads
  • You must be logged in to reply to this topic.