RNA Isolation - Integrity of RNA (Apr/11/2005 )
I am isolating total RNA from CaCo2 cells. In agarose gel it always appears the 5S-tRNA band too intense (about 5 times the intensity of the 28S and 18S rRNAs). However the ratio between 28S and 18S is close to 1 and there is not considerable smearing under the 28S and 18S. So I would like to ask:
1) What is the optimal ratio between the 28S and 18S RNAs witch indicates that the RNA is not degradated?
2) If the 28S and 18S are normal, how could I interpretate the excesive amount of 5S-tRNA? is RNA degradated or what?
3) Are CaCo2 difficult cells for RNA isolation?
I ve tried many ways to improve the extraction but no matter what I do the problem continues. thanks for any help.
don't check the integrity of your RNA by running the gel, because it's time-consuming. just use NANODROP to check the quality of RNA. i don't think Caco2 a difficult cell line for RNA isolation. you'd better use RNAeasy mini kit from QIAGEN. it works very well and you will never fail. good luck.
1- Optimal ratio between 28S and 18S should be right around 28/18, which is 1.56. They come from ribosomes and if nothing happened to them the ratio should be 28/18.
2- 5S RNA should not be that intense. I can think of maybe... you are leaving too much supernatant when you are transferring it after the centrifuge? During centrifuge, RNAs line up based on their weight in the centrifuge tube. So 5S are all on the top of the liquid. then 18S, then 28S. So if you leave out some liquid it will contain lots of 28S then 18S. This also explains your 1:1 ratio between 18S:28S
3- Caco-2 cells are not difficult for RNA extraction. I use them too and I get great RNA everytime.
Hope these are helpful.