RNA labeling with Biotin - (Aug/26/2011 )
I really need your thoughts and ideas about how to go on labling my RNA with Biotin.
So my RNA is a hairpin structure that was synthesized by me and gel purified; it's composed of 72 nucleotides and it has tri phosphate at the 5 prime end.I need to lable it with biotin so i can move on to SPR technique.
here are the steps that i follow:
1) removing tri-phosphate from the 5 prime end
my RNA concentration in a 100uL rxn is 1uM
Alkaline Phosphatase buffer, Alkaline phosphatase enzyme
total volume rxn:100 ul
incubating the rxn f0r 25 minutes at 37C then increase the temperature to 50C for 15 minutes
prepanol (equvelent volume) and sodium acetate (1/10 of the volume)to preciptate my unphosphorlated 5 prime end RNA
add equvlent amount of 70% ethanol and centrifuge again
dissolve the pellet with minimum amount of ddH2O
2) adding PNK (T4 Enzyme), PNK buffer, ATP-gama Sulfur (0.4 mM in the final volume) and my 20 uL dissolved RNA
up to 50uL rxn
leave the rxn in an incubator for an hour at 37C then raise the temperature to 70C for 10 minutes.
remove any excess unreactive ATP-gama S using spin colume.
speed vac. for an hour or so
3) adding my EZ-Link idoacetyl-LC-Biotin
o.4 mM in a final volume rxn, K3PO3 buffer at PH 8.0, my previous RNA volume
4) loading in a 10% denaturing gel
Ok, so by following this protocol with my hairpin RNA strand, i'm not able to get the supershif. so I was curious if my reagents (i.e. ATP-gama-sulfur, PNK enzyme etc) are inactive and I decided to follow the same protocol using single stranded DNA strand (28 nucleotides) and the results were positive that is i saw the supershift in a 20% denaturing gel.
I'm just confused about why my RNA is not getting labeled......which step of the above is not happening....( i took few micoliters of each step's product to examine it using MALDI, but the MW of my RNA itself is too large and MALDI is not detecting it)
I also read that before adding my PNK enzyme and ATP-gama-Sulfur, I should heat my unphosphorlated RNA to denature it and then cool it immedatily on ice. That way PNK will recognize the 5 prime end easily because remember that my RNA is a hairpin structure so adding that gama sulfure to the unphosphorlated 5 end would be diffcult. So i'm gona try that today and hopfully it will work.
if it doesnt work I need to think about new ideas on how to lable my RNA and maybe someone can help me with my problem by replying back to this post.
I would be thankful for any thought or idea from you guys
I think that Pierce Biochem (now owned by Fisher) makes a 3'end biotinylation kit. The chemistry is basically the addition of a Biotin-CTP to the 3' end of the RNA using T4 RNA ligase. You may be able to also just do this on your own by purchasing T4 RNA ligase and Biotin-pCp separately.
Best of Luck!