What is the source of your RNA sample? Is it total RNA from plant material, animal material? Did you OD your sample before you precipitated it? Usually contamination with proteins and carbohydrates will make the RNA pellet hard to dissolve. The OD260/OD280OD measurement will tell a lot about the quality of your RNA. Pure RNA has an OD260/OD280 ratio of ~2.0. Low ratios could be caused by protein or phenol.
You can dissolve the RNA pellet with warm RNAse-free water or TE buffer (50-65 °C), - do not heat the pellet. As Joanna already mentioned, you must verify the quality of your RNA.
its total RNA from plant material jojoba, no I did'nt take the OD value, as the pellet was not dissolving
You have not mentioned which technique has been used to isolate RNA from jojoba. If the RNA is not contaminated with protein or carbohydrates; you better take notice that all traces of salt has been removed. You can add a small amount of NaOAc or NaCl solution, may be a 20th volume of 3M say, then add 2-3 volumes of 95-100% ethanol and treat it like a standard ethanol precipitate. If you don't eliminate the salt, the RNA will be next to impossible to dissolve.
I used guanidium thiocyanate isolation process and used 3M NaOAc also for precipitaton along with isopropanol.
Hi, Himanshi. I am not expert in RNA extraction from plants, but I had some troubles to extract RNA from insect embryos with high amount of fat in the egg yolk. You can search especific protocols for plants at springerprotocols. You can look these:
http://www.springerprotocols.com/cdp/search/searchResultPage?text=extraction+rna+plant&submit=Go
@Marcio Fontenele.. thanks a lot.. but i cannot access these protocols, can you send me the pdf versionif possible.
HI Himanshi
The pellet may be due to several contaminants as mentioned by researchers above. .....if you follow some of the approaches mentionde above to increase the solutbility and still cannot completely dissolve it..you can do the following..
Some fraction of your total RNA may already be in solution ...and give a brief spin to pellet out the insoluble contaminants and take the OD of the supernantant (or run it on a gel) to see if sufficient RNA is present in the solution and if it is sufficient for your downstream processing...go ahead and use it..
@Ajay Saini...I did this already and run this supernatant on gel, but i could'nt anything...
Along with the contamination, if RNA sample is not properly dried, it cant dissolve. Let the sample properly dried before adding TE for water and wait for 20 min to dissolve it.
I used this procedure and had massive success with RNA isolated from plant (tomato seedlings) roots.
Precipitate with isopropanol, centrifuge and wash with 70% EtOH, redissolve in warm RNase free water.
Assumption: you have undissolved RNA in RNase free water and stored it in -80 degrees, and probably wondering what next....
1. Add ~500ml isopropanol to your the -80 frozen RNA sample (the ice will melt immediately, leaving you to stare at the undissolved pellet).
2. Incubate at -20 for 1 hour
3. Mix by inverting tube; spin at ~15000g for 10min; carefully discard supernatant
4. Wash pellets in ~ 750ml EtOH (room temperature) three times; air dry (with tubes facing diagonally downward on clean tissue paper)
5. Dissolve in warm (~55 - 60 degrees C) RNase free water
6. Incubate at room temperature for ~3 min; then keep on ice (if you are going to do spectrophotometric reading immediately, otherwise store at > -70 deg C.
7. I have had success not only with improved 260/280, 260/230 ratios (from between ~ 0.76 &1.05 to 1.79 to 2.34), but also very good dissolution and high concentration of RNA.
Sonali Sangwan: sorry for the typo; I meant ~500 microliters (uL) of isopropanol. Also < -70 deg C (not > -70 deg C).
Remember the amount of isopropanol will depend on the capacity of the tubes containing your samples; I had added the 500 uL on to each of my 50 uL samples frozen in 1.5 ml tubes at ~ -80 deg C.
Hope that helps.
@henry awika thanks for the protocol. I am having the same problem of undissolved pellet. I have tried your protocol. But some percentage of total RNA pellets are still undissolved. What can I do further?
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