Thanks to all of you for your help, finally I had to precipitate the samples after the miRVANA isolation to ensure that they were all free from phenol and after the precipitation step they were all perfect in the nanodrop and Bioanalyzer.

mirvana is a good kit for small RNA isolation. The 160/280 ratio tells the purity of the RNA, ranging from1.8-2.2 in general. If you worry about the quality, you might like to check it with Bioanalyzer which tells you the integrity as well.

Hi, I used the recover all kit from ambion when I did some runs for miRNAs on the open array. That worked well. Never tried the mirvana kit though. The 260/280 >2 is good, the 260/230 is too low. Look up the trouble shooting. I usually see 1.5 or higher. Good luck!

Thanks Yongjun. I have run my samples in the tapestation, which is similar to the Bioanalyzer and it says that "not marker is detected" and I also get a strange profile with many bands all over the lane. Maybe there is something in the samples that is degrading the marker?

I have personally used this kit before and it worked well. I find the most important step is when you take the top phase after adding the acid-phenol, traditionally one would try to take as much as you can, but if could you aspirate a little less you will sacrifice some of the RNAs but the purity will be very good. I also find increasing the centrifuge time also helps, I usually do around 5-10 mins. What kind of array are you looking to do? qPCR or Fluoresce probe using hybridisation?

p.s. make sure your column is completely free of ETOH at the end, even if you have to do one more spin (1min) because having residual ETOH really effects the quality of your RNA. Iif your yield is low, try heating the water used for eluting the RNA and reduce the volume to around 25ul. 95 oC is perfect but with this volume you might find evaporation an issue, I tends to use 65oC and stand for 5-10 mins before last spin (for collection)

The low 260/230 ratio (0.27) indicates that the eluted sample is still having the phenol as contaminant (the miRVANA kit has Acid-Phenol:Chloroform extraction step). You have taken 50mg tissue but used 600uL of lysis buffer (instead of 500uL, i.e. 10 volumes). In that case, try to do one more washing step and dry the spin column with additional centrifugation. Then elute the sample in new collection tube. You may also check the sample quality on bio-analyzer using Small RNA Analysis Kit.

Your low 260/230 ratio is due to high absorbance at 230 nm, which suggests contamination of your preparation, probably with phenol and thiocyanate salts. As Kelvin pointed above, during the phase separation step is very important not to take any of the lower (organic) phase. During separation of the aqueous phase, you should keep your pipette tip aways at the top of the aqueous phase and aspirate gently/slowly, so you do not disturb the organic/aqueous interphase. Even if you are aspirating a large aqueous volume, I recommend using a lower capacity pipette tip (so you can handle the aspiration more gently, e.g., a 200 ul pipette) and aspirate a few times as needed. Always leave some aqueous phase on top of the organic phase (let's say about 1 mm high, more or less as you feel comfortable), so you avoid getting too close to the organic phase. You could try to clean this particular sample with an additional chloroform (chloroform:isoamyl alcohol 24:1) separation step and precipitation of your RNA, but the same principles operate during separation of the aqueous phase from the organic chloroform phase to avoid carrying chloroform. If the OD 260/230 ratio continue to be a problem for your future mirvana separations, you could always include the chloroform separation step right after you separate the aqueous phase from the phenolic phase. One additional step that will lower your yield a bit, but your prep should be cleaner. And remember to be gentle aspirating those aqueous phases and not to take any of the organic phase or interphase (stay away from the interphase!).

I personally have a very good experience with mirVana kit. Do you perform the total RNA isolation or enrichment procedure? I would suggest longer centrifugation time, to my experience any centrifugation shorter that 30 sec resulted in remaining liquid above the filter.

Ravi and Yuri are correct, separating the phases after organic extraction is the problem. Believe me you'll have enough RNA from that size tissue. Be conservative and avoid the interface. Or be conservative and then add smoe buffer back to the organic phase/interface, vortex, and repeat the removal of the aqueous phase. You can then pool the aqeous phases, adjust the salt and alcohol and run it all over apurification column.

Hi Melissa,

I work on microRNA as well, and I tried mirvana paris, trizol and other kits. Differently from the other I didn't like this kit, you easily have phenol contamination (as for trizol) and I didn't like the enrichment part. For microRNAs I don't enrich my sample but I prefer to isolate total RNA, but this is a suggestion. Anyway the ratio 260/230 is very low, and the contamination from a component of the lysate component such as guanidine thiocyanate could be the problem. In this case try to wash better sample or and column, and as the other already said, avoid the last part of acqueous phase.

Good luck

Claudia

I also had some problems with Bioanalyzer profile. This often a problem of electrode or set date kit (when marker is absent). For the odd profile, it's can be electrode problem and I suggest you clean them with a fine brush and drying well.

Hi Melissa, I have used the kit and have obtained a good yield with acceptable 260/280 ratios. Are you careful to pipette out only the top layer after the phenol step. Disrupting the layer might increase phenol contamination. Also, I never enrich for microRNA population but rather extract total RNA. It is much better for arrays.

Hi Melissa,

If the separation of the phases might be an issue, try to use the PLG tubes (http://www.5prime.com/products/nucleic-acid-purification/organic-nucleic-acid-extraction/phase-lock-gel-.aspx), they have a gel that will stay in the middle of phases, so you can take all of your aqueous phase. We have used them in RNA extractions and worked smoothly.

BTW, best regards form your former department. :)

Thanks to all of you for your help, finally I had to precipitate the samples after the miRVANA isolation to ensure that they were all free from phenol and after the precipitation step they were all perfect in the nanodrop and Bioanalyzer.

Hi Melissa,

Finally, what did you use for the precipitation? I have the same problem with the low ratio 260/230 and when I use the RNA clean and Concentrator kit from Zymo Research I lost the most of sample. Some idea? Thanks in advance!

Hi Maria,

I did a normal RNA precipitation using one volume of Ammoninum Acetate 5M and 8 volumes of ethanol, -80C 30 min, centriguge at 4C 16000 g 30 min, a quick wash with 1 ml ethanol 70%, centrifuge and resuspend in water... and it did improve the ratios and the open array worked after that. I wish you good results!

Thanks SO much!! I got it! I hope to have the same good results than you :-)

Maria, just to make something clear... because I am not sure it was when I wrote it before, if you have 100 ul of RNA, add 100 ul of Am Ac, mix and then add the 800 ul of ethanol. Hope it works ;-D

Dont worry I understood you well but anyway thanks so much to explain me again ;-)

Excellent i got good results with this purification after mirvana isolation :)

I am getting different results with ethanol precipitation after miRNA isolation with mirVana kit. I got a couple of samples with good 260/230 ratio but most of samples still have not acceptable 260/230 ratio - less than 1.1-1.5. I tried the second precipitation and sometimes I get even lower 26/230 ratio than after the first ethanol precipitation. What could be a reason for that? Thanks.