How pure are my RNA samples - (Dec/19/2006 )

Hi
I've isolated total RNA from animal tissue by TRIzol method. I'm getting good A260/2801.8-1.7 but my A260/230 ratio is not good they vary from 0.81- 1.61. Does this indicate poor quality of RNA. I'll be later on purifying RNA by Qiagen's kit.
Why this problem is arising?
Any answers...
Bye!

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it may indicate phenol contamination. do another chloroform extraction.

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I have already extracted twice with chloroform, should I increase it to 3 or 4 times. As I have already isolated RNA, will this contamination of phenol will go with qiagen kit?
any suggestions regarding the efficiency of this kit?

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i haven't used the qiagen kit but it most likely will clear the rna of phenol contamination (if phenol is even used for the extraction).

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May be that u r not pipetting out the separation phases properly.. Pipette out only half of the upper phase, followed by the two volumes of ice cold isopropanol treatment, followed by 1 hr incubation at -20C.

I hope this is helpful.. Good luck..

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FYI, I've gotten low 260/230 ratios using Qiagen's RNAeasy kit as well. The low values may not be phenol, but are more likely salts, since Qiagen's kit is phenol free. Reextracting your samples may help, but you are likely going to lose yeild. RUn it out on a gel, does it look degraded? is the sample of low concentration? i've noticed that low 260/230 is often going hand in hand with low concentration samples... i think its due to less RNA, but the same ammount of residual buffers, etc that might get washed out. However, if you are using the rna for a RTPCR reaction, the cDNA synthesis buffer usually eliminates pH and salt issues from your RNA samples. So, in generall.. i dont worry too much about 260.230 myself.

hope this helps.

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you need to precipitate with isopropanol or buOH (try to avoid etoh that loose greater mounts of nucleic acids, or precipitate but allow -80 for 2H) and let better dry youy samples.
that should be sufficent

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