I extracted RNA from human nodal tissue using trizol/chloroform extraction followed by isolation with a Qiagen kit. The RNA concentration of all cases was good (>200ng/µl) but the 260/230 ratio was low in most cases (= 2) of the cDNA samples are very good for all cases. However, is it possible that somehow the cDNA does not reflect the original RNA because e.g. some RNAs were less efficiently reverse transcribed than others due to phenol contamination? In other words: can I use the cDNA I have now to do QPCR?
230 is usually guanidium: 270 is phenol. Still, guanidium is also very toxic to downstream reactions, so the question remains valid.
A lot will depend on
A) how much lower than 1.5 your 260/230s were (below 1.0 is bad, 1.46 is...potentially manageable)
B) how much RNA you use for cDNA synthesis: guandium inhibition is concentration dependent, while 260/230 is obviously ratiometric. The more RNA you use, the more guanidium you're contaminating your reaction with. Using low amounts of RNA (say 200ng) will thus permit viable cDNA synthesis with worse 260/230 ratios than would using 2ug of RNA.
You're using reference genes (I hope) so one quick check would be to simply measure reference gene signal in all your samples (both those with good 260/230s and those with bad ratios): if you get consistently lower signal from those prepared from dirtier RNA, then you should probably reclean those RNAs and prepare fresh cDNA.
On balance, I would say the likelihood of salt contamination interfering with reverse transcription of specific RNAs but not others is....low, but it generally pays to play it safe even so.
I've had trouble with phenol contamination in the past. Unless you estimated the RNA concentration using a method other than UV spectroscopy I wouldn't trust the RNA concentration you measured, for one; too much overlap in the absorption spectra. Phenol and guanidium (particularly the latter) can be fairly destructive, but if they're at sufficiently low concentrations you may get away with it.
I've found that multiple chloroform:isoamyl steps following the single phenol/trizol treatment may be necessary to remove lingering phenol and other contaminants in some cases. Three equal volume chloroform extractions will usually do the trick.
Thank you very much for your replies! To be sure and to ease my mind I will redo the extractions.
Dear Julie Morscio
Considering the opinions above I can only suggest one more possibility.
Even with the great risk of losing precious material, I would perform another precipitation step, just with ethanol.
Only then you'll be sure about the quantities you measure.
Good luck
Dear Dr. Gonçalves,
Thank you for your suggestion! Could you tell me how I would have to do that? I have my RNA suspended in 40µl RNase-free water.
Hello,
I would suggest you to perform a standard RNA precipitation protocol.
For example:
- add 60 ul RNAse free water to your sample (total of 100 ul)
- 10 ul 5M LiCl
- 300 ul Ethanol 100%
Leave it in the -20º for 30 minutes (when I have time I leave it one hour or during lunch time). Some people prefer using the -80º but considering all the precessing steps it doesn't make much sense.
Centrifuge at 4ºC during 30 minutes at 13'000 RPM
Remove the supernatant and add 250 ul 70% ethanol
Centrifuge another 10 minutes.
At the end suspend with 40 ul again.
You might lose half of the RNA but you'll have good quality and non-contaminated material.
To store it I really recommend you to keep it at -80ºC.
Good luck
Hi Juli
There are two different things you should think of.
The first is that you can't in anyway (unless u run it on gel actually) measure the concentration of cDNA. It is a very common mistake, The reason is that your tube is full with dNTPs in a very big excess. The way in which you quantify the concentration of your cDNA is by calculating how much RNA you entered to the reverse transcription reaction.
Now secondly for your question itself. The problem with phenol is that it hampers the reverse transcreptase. What you should do is run all your samples on real time and check the levels of your house keeping genes. Compare between the samples and make sure that each of the house keeping genes go up in relative close cycle. (Compare the samples not between the house keeping genes off course).
If they all go up around the same cycle then you dont have a problem with your RNA or cDNA. Just make sure you load the same concentration of cDNA to the real time calculated from the RNA concentration.
I hope it helps
Amitai
Dear Juli,
I do not know how you have managed to get PCR products with phenol contamination, however, in your posting you never mention of any control that you should have run with this experiement. However, to be on the safe side, I will reprecipitate my RNA using standard salt and ethanol protocol and then measure the amoubt of RNA if possible on Agilant RNA quantification appartus to know realy what you have in your eppendruff tube. The mere of the fact that you have gotten phenol contamination, sorry my frankness, it means you have not taken the proper care and extra care to work witn RNA which is very fineaky to work with.
Regards,
GCK
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes