miRNA cincentration? HELP needed! - (Jun/05/2008 )
Hi everybody! I've got a BIG problem here. I'm using miRVANA PARIS kit to isolate miRNAs from various liquid samples. It's the only kit working so i can't switch to another kit. the problem is that i obtain a fair amount of miRNAs (either total RNA or miRNAs i can have both) but concentration is poor (5/6 ngs/ul). I can pool many samples but the result is 500-600 ul of RNA at 5/6 ng/ul. I tried eluting with less water but to no avail, so i need to concentrate mi RNA.
precipitation with either sodium acetate or butanol did not work well (result few ul of water, 5/6 ng/ul!).
I also tried speedvac, but i still loose a lot of RNA (sometimes i reach 20-25 ng/ul in 50 ul, often less, 9/10 ng/ul).
Last thing i've tried is qiagen RNeasy MinElute kit with a modified protocol to concentrate miRNAs from my eluted RNA (the protocol has been given to me by qiagen technical service). The result is quite unhearting, i lost every single ng of miRNA!
can anybody help me? any suggestion?
somebody told me to try speedvac using silicone coated tubes to avoid RNA sticking to the walls of the tubes, but i have no clear idea on how to do that.
Thanks everibody for ANY kind of suggestion.
Fizban
EtOH precipitation usually work well.
Add three vol of ethanol and 0.1 vol of 3M Na acetate pH 5.2 (or 0.1 vol NaCl 5M) and incubate overnight at -20. If the conc is low add a small amount of glycogen. I use 5-10 micrograms in a total vol of 2 ml. Spin at 14000 for 30 min at 4°C, wash with 80% ethanol.
I use siliconize4d tubes from sigma, but with some concerns. They're not guaranteed RNase free and if autoclaved sometimes turn opaque, so I just wash them with 80% etOH before use.
Add three vol of ethanol and 0.1 vol of 3M Na acetate pH 5.2 (or 0.1 vol NaCl 5M) and incubate overnight at -20. If the conc is low add a small amount of glycogen. I use 5-10 micrograms in a total vol of 2 ml. Spin at 14000 for 30 min at 4°C, wash with 80% ethanol.
I use siliconize4d tubes from sigma, but with some concerns. They're not guaranteed RNase free and if autoclaved sometimes turn opaque, so I just wash them with 80% etOH before use.
i've already tried Na acetate to no avail.
I'll try with siliconized tubes.
thanks anyway
Fizban
Isn't the PARIS kit for RNA, DNA and protein? Have you tried the regular mirVana kit jut for total RNA? I know you say that you can't change to a different kit, but the columns used might be the same (only different wash buffers different?). My PI said to me a while ago that these kits that try to give you all the different sample types often end up not giving you much of either...
How do you quantify your RNA? Are you sure that you actually have RNA in your sample? Since the reading is so low, maybe it is just background and you're not actually concentrating your RNA because there isn't any there to concentrate? Such a low reading is ok, just about, for a nonodrop reading, but wouldn't trust such a reading on a regular spectrophotometer.
Good luck!
How do you quantify your RNA? Are you sure that you actually have RNA in your sample? Since the reading is so low, maybe it is just background and you're not actually concentrating your RNA because there isn't any there to concentrate? Such a low reading is ok, just about, for a nonodrop reading, but wouldn't trust such a reading on a regular spectrophotometer.
Good luck!
Hi,
yes, PARIS kit is for DNA, protein and RNA. Ambion technical service told me to try the "regular" mirVana kit like you, but it does not differs from paris kit when speaking about liquid samples.
And yes again. I'm actually using Nanodrop to quantify my RNA and RNA IS actually there, since i'm able to amplify miRNAs from my samples and negative controls remain negative.
I would not mind about concentration if i hadn't to use the new low density arrays from applied biosystem. you need to use at least 100ng of RNA for a single RT reaction, but only 2 to 5 ul in 10 to add your RNA.
that's a limit that i need to overcome in order to start my project, so i really need to concentrate my RNA.
Thanks for your answer anyway, i'll manage something, i'm not one to surrender easily!
fizban
I am using that array. To prepare the loop specific cDNA you need to add 19.25 mcl of water to the mix..you can use that volume in your total RNA volume instead of in the mix. Also, you can talk with ABI guys and since you have a 62.5X dilution of your cDNA to prepare the samples for the card, probably they know how low can you go in that second dilution
Good luck