I am doing RNA extraction using Trizol Reagent from Invitrogen. I got rations A260/280 1.60 and A260/230 1.19. Is it possible to clean up my samples and increase my rations? Is it useful to precipitate my RNA with ethanol 75%?
Dear Aletheia,
For RNA isolation by Trizol method, there are two steps to be taken care off to get good ratio of A260/280 and A260/230.
1. Collect the upper aqueous phase carefully without disturbing the interphase or phenol-chloroform phase. If necessary leave away some amount of aqueous phase at the lower side so that you are not disturbing the lower layers.
2. Two successive washes with 75% ethanol after isopropanol precipitation of RNA would be helpful and takes care of your ratios.
Good luck with your RNA isolation.
Hi, Alethenia, If you have high absolite value (A260) ypu can try ethanol precipitation. However, if your A260 is very low, you have a risiko to lose RNA. Again, if you have high A260 (high RNA concentarion), you can try to process next step (cDNA). In that case it may work, but I can noit be 100% sure.
May be better to isolate new RNA. Good luck!
Hi Althenia,
A column cleanup is likely to help you with this I think as it is a sign that there is potentially too much phenol or ethanol carryover. A column clean up will definitely help to improve the ratios and remove excess phenol and ethanol.
Hi Althenia
We routinely take the clear upper fraction from the Trizol extract and put it down a QIAGEN RNeasy column. Wash with the kit buffers and elute with 30 - 50 ul RNase free water depending on how concentrated you want your prep. Should get good ratios pretty quickly.
Good luck
Claire
Aletheia you can use RNEasy columns from quiagen and use DNAse in your RNA preps as well to purify your RNA samples, quiagen RNEasy columns can hold up to 100 micrograms RNA so it should be fine with you if you are using the RNA for real time PCR, but if you will be using northern blots, use multiple columns for the same sample and if it is diluted then use speedvac to evaporate water and concentrate it again using DNAse/RNAse free water. Make sure not to exceed 55 degrees when doing that otherwise you might affect your RNA stability. This temperature should also help in cDNA preparation from RNA at the beginning of your cycle as it will help you reverse transcribe RNA from secondary structure as well.
Good luck
Try to measure your RNA sample on another UV spec, just to be sure 100% that it is your sample that has the problem. Old UV lamps tend to give low ratios, I have experienced that before.
Cheers
Anwar
yes,but did not mentioned the ratio of absorbence if it comes 1.6,1.5,1.7 that is slight below 1.8 and your experiment is on rael time pcr for quantification then it give good results,otherwise there is always risk of wash out.
But if quantity is also good then i will suggest u for precipitaion with ET-OH.
Hi Alethia,
One important factor influencing the A260/A280 ratio is protein contamination and this is why you are concerned about cleaning up your samples. You have some answers for this above.
Another important factor affecting A260/A280 ratio is the pH of the solvent in which redissolve your RNA pellet after extraction. If you use water or TE buffer pH=8, the same sample will have a very different A260/A280 ratio. Often commercial kits use TE to resuspend the sample and this will typically increase your A260/A280 ratios. I observed ratios of 1.7-1.8 in water and 2.0-2.2 in TE with the same previous extraction steps.
Jean-Charles
I am isolating RNA through Trizol extarction. I have got low A260/A230 ratio. Although A260/A280 is fine. Should I wash my RNA with 75% ethanol or use qiagen RNeasy at this stage?
Might be you have trizol contamination in your RNA. Read this article about it. I hope it will be helpful.
http://www.ncbi.nlm.nih.gov/pubmed/19146820
Hi Aletheia,
You are not the only one experiencing the low 260/230 from Trizol extraction. it is simply due to the residual contamination from phenol. Trizol is an excellent method but as all others recommended you may have to go through a clean-step (column based) that you have to pay an extra cost. As a long time lab researcher I knew about this problem and developed the RNA kit that can purify large and small RNA without using phenol. As you expect since no phenol, the 260/230 is better from even challenging plant samples such as grape, strawberry, pine needle etc.... http://norgenbiotek.com/display-product.php?ID=53
Dear Aletheia,
For RNA isolation by Trizol method, there are two steps to be taken care off to get good ratio of A260/280 and A260/230.
1. Collect the upper aqueous phase carefully without disturbing the interphase or phenol-chloroform phase. If necessary leave away some amount of aqueous phase at the lower side so that you are not disturbing the lower layers.
2. Two successive washes with 75% ethanol after isopropanol precipitation of RNA would be helpful and takes care of your ratios.
Good luck with your RNA isolation.
Hi Aletheia,
Treat your ground sample initially with Extraction Buffer containing 100mM Tris HCl , pH 9.5, 150mM NaCl, 0.5% ϐ Mercaptoethanol (add fresh). Collect the supernatant for trizol extraction. Its improves the purity of RNA dramatically in Arabidopsis seeds and siliques.
My bloody experience: Wash the RNA pellet with at least 3x 75% EtOH at RT. Importantly, when adding EtOH, remember to roll and invert the eppendorfs so that it can wash any salts binding to the tube wall. You can gently resuspend the pellet when washing by flicking the tube. Washing EtOH at RT can remove the remanining
salts more quickly than washing with chilled EtOH, because the chilled EtOH make the pellet more solid, not contacting the washing buffer well.
In my own experience doing RNA extraction with Trizol what is very important is doing 2 washes with 70% ethanol. Use also room temperature Isopropanol to avoid salt precipitation and do not let precipitate overnight ( I do not know if you did that), better only 1 hour. Ratios 260/230 were low before, now they are very good. Good luck!!
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