Has anyone freeze-dried their miRNA samples (in a separate small RNA fraction, not in total RNA) in order to increase the concentration? Does it affect the RNA integrity?
Hi Stefanie,
I have used at least 3 methods to concentrate RNA/microRNA:
1) Speed Vac. Had similar experience as Matteo (no clear signs of deterioration)
2) Qiagen columns: the mini/micro kit for microRNA isolation contain columns that allow elution in as little as 14 ul, but to maximize binding of the microRNAs you have to use a higher EtOH concentration to add to your aqueous sample before column binding, usually you will mix it with 1.5 fold of 100% EtOH. So the concerns raised by Sander can be easily overcome. You can buy the columns and the rinsing buffers alone, without purchasing the whole kit.
3) EtOH precipitation and re-elution in smaller volume (higher yield than column based, if performed well). Make sure you add glycogen, because it is the only way you will actually see a pellet after the first precipitation.
Hope this helps!
I did speed-vacuum treatment of total RNA containing miRNAs, also at 37 C, thus concentrating the sample without any clear bad outcomeon the miRNA integrity as evaluated by RT-qPCR. But it was total RNA and it was the opposite of what you want to do...
I usually use cleanup kits by Qiagen, that give upto 14uL of highly concentrated miRNA.
Dear Mustafa I have problem with low miRNA concentration as well. Do you extract RNA using miRVana or Trizol? And could you provide a catalogue number of this Qiagen kit that you are using to concentrate your miRNA?
Be careful when using Qiagen kits or any other silica-based kit for that matter. They have a size cutoff (usually ~ 50 nt) and miRNAs might not bind efficiently. You might get a much higher yield if you would do the old fashioned EtOH precipitation with glycogen as carrier.
Hi Stefanie,
I have used at least 3 methods to concentrate RNA/microRNA:
1) Speed Vac. Had similar experience as Matteo (no clear signs of deterioration)
2) Qiagen columns: the mini/micro kit for microRNA isolation contain columns that allow elution in as little as 14 ul, but to maximize binding of the microRNAs you have to use a higher EtOH concentration to add to your aqueous sample before column binding, usually you will mix it with 1.5 fold of 100% EtOH. So the concerns raised by Sander can be easily overcome. You can buy the columns and the rinsing buffers alone, without purchasing the whole kit.
3) EtOH precipitation and re-elution in smaller volume (higher yield than column based, if performed well). Make sure you add glycogen, because it is the only way you will actually see a pellet after the first precipitation.
Hope this helps!
THE BEST FOR YOU IS USE TRIZOLE FOR EXTRACTION AND CLEAN WITH MinElute Reaction Cleanup Kit (50) 50 MinElute Spin Columns, Buffers, Collection Tubes (2 ml) 28204 QIAGEN. Probably this will help you.
Hi Karolina,
We use miRneasy Kit Qiagen (Cat No 217004) http://www.qiagen.com/products/rnastabilizationpurification/microrna/mirneasyminikit.aspx#Tabs=t0
Also if u are really interested in concentrating it then you can use one of the clean up kits, they would be mentioned in the protocol book that comes along .
http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasyminelutecleanup.aspx
I have to expression profile different miRNAs in clinical samples (most of which are with low GC content). I was planning to use TRIZOL as it has given me good yields (upto 800ng/ul from 3mg tissue without using any carrier or modifications in protocol) but now I am thinking should I continue to use TRIZOL after reading the following retraction of paper
http://retractionwatch.wordpress.com/2012/07/13/group-retracts-microrna-paper-after-realizing-reagent-was-skewing-results/
Dear Azeem, just to be sure: did you really mean: ..."but now I am thinking should I continue to use TRIZOL after reading ..." or did you want to say:....but now I am thinking should I to use TRIZOL after reading ....?? Very best wishes and good luck, Wolfgang MUSS, Salzburg, Austria
@ Dear Wolfgang, Ah yes, it was like questioning myself =) "Now I am thinking should I discontinue to use Trizol". as I have managed to obtain good yields with Trizol. I will be using mirvana kit next week for RNA extraction from tissue samples so lets see how it goes :)
Dear Azeem, thank you...(I admit that I am following this question as a non-User of such methods but just am interested in those techniques. And yes, when reading your text passage a third time I got it! (it was like questioning yourself =) "
best regards, Wolfgang
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