RNA quality - (Oct/08/2010 )
I've extracted mosquito RNA using kit and hot-phenol method. It seems to me that all my purified sample were of low quality since 28s:18s ratio is very low. Are they good enough for downstream application such as RACE and qPCR?
http://img188.imageshack.us/img188/2593/rna.jpg
2nd lane is my kit-purified while the other 4 are hot-phenol purified. Ignore the ladder please.
would be great to have a marker ...this could be everything in fact.
Normally, a 2:1 ratio of 28S to 18S is considered good ...but this does not mean that a sample with another ratio fail in qPCR experiments. It always depends. If you do your assays repeatedly, you should try to improve your extraction method so that your RNA quality is constant throughout your experiments.
Here is a good review on that topic.
Regards,
p
I am not sure if you are showing us denaturing or native gel. I did not know people check 28s:18s ratio. I just learned.
If what you are showing us is the native gel, I do not think you can tell the ratio correctly. Ethidium bromide will only bind to double stranded RNA. If your gel is native, then intensity of stain would be depended on RNA strands that form double stranded structure. Therefore, you should not use native gel to compare ratio. You do not know if 28s and 18s make dsDNA at the same rate. In my opinion your RNA looks nice and you are ready to go to next step.
I hope this make sense. Good luck.
http://www.bioprotocols.info/
pDNA on Mon Oct 11 20:42:07 2010 said:
would be great to have a marker ...this could be everything in fact.
Normally, a 2:1 ratio of 28S to 18S is considered good ...but this does not mean that a sample with another ratio fail in qPCR experiments. It always depends. If you do your assays repeatedly, you should try to improve your extraction method so that your RNA quality is constant throughout your experiments.
Here is a good review on that topic.
Regards,
p
chromatin on Tue Oct 12 01:09:09 2010 said:
I am not sure if you are showing us denaturing or native gel. I did not know people check 28s:18s ratio. I just learned.
If what you are showing us is the native gel, I do not think you can tell the ratio correctly. Ethidium bromide will only bind to double stranded RNA. If your gel is native, then intensity of stain would be depended on RNA strands that form double stranded structure. Therefore, you should not use native gel to compare ratio. You do not know if 28s and 18s make dsDNA at the same rate. In my opinion your RNA looks nice and you are ready to go to next step.
I hope this make sense. Good luck.
http://www.bioprotocols.info/
That was a native gel. I wish i have the marker but all RNA marker in our lab are expired (2005). My boss said he is not going to buy because i am the only one using it.