Need help with extracting miRNA from human serum using mirVana kit - (Jan/08/2012 )
My colleague and I are having difficulty extracting miRNA from human serum using the Ambion mirVana miRNA isolation kit. We have used other methods with some success but the mirVana kit is really giving us some trouble. We have tried optimising the protocol but it seems we cannot get any miRNA at all. Can anyone shed any light as to where we might be going wrong?
This is what we have tried:
<*>Blood is collected, clotted and serum removed. Serum stored at -80C until needed.
<*>500uL serum is used for miRNA isolation. Two volumes of lysis buffer (in the ambion kit) is added to the serum. Note, this is done in two eppendorf tubes (250uL serum + 500uL lysis buffer in each tube). Incubated for 5 minutes on benchtop.
<*>Homogenate additive added (1/10th volume - so 75uL) and incubated on ice for 10 min.
<*>Phenol/Chloroform added and centrifuged for 10 min at RT. Interphase is compact and well defined.
<*>Aqueous phase removed (roughly 750uL) and 1.25 volumes of 100% EtOH added.
<*>At this stage, both tubes for each serum sample are combined into one filter cartridge.
<*>Wash buffers are put through the filter as per protocol.
<*>Elution: elution buffer is heated to 95deg. We have tried eluting with 100uL as per the protocol; also, eluting with 30uL and eluting with 2 x 50uL.
When read on the spectro (we don't have easy access to a nanodrop) we get consistent readings of 0 for concentration and 260/280 ratio. Our spectro is old and we dilute the elution down to 1/50 for it to be read. We have also tried diluting down to 1/25 but if we dilute less then we risk losing our extracted miRNA. We dilute in ultrapure water.
I've been reading these forums looking for some ideas and I know some of you have been successful in extracting miRNA from serum. Any help would be greatly appreciated. Thanks.
Have you tried a positive control for the spec, in order to be sure you are able to see an absorbance if it is present?
Hi, thanks for your suggestion.
We ended up solving the problem by accessing our nanodrop. The miRNA yield turned out to be on the ng/uL scale, rather than the ug/uL scale so our spectro was having serious issues picking anything up. Having to dilute down the sample to read it on the spectro was also throwing more problems to account for.