Antisense oligonucleotide application

Prepare ASO aliquots:

1. Order DNA Oligos from Integrated DNA Technologies (idtdna.com)

a. The first and last five bases should be 2’O-Methyl RNA bases, standard destalting is fine

b. Use published sequence or design your own

c. Ours were adapted from DeVos et al., 2013

i. Tau ASO sequence: 5’-mA.mU.mC.mA.mC.T.G.A.T.T.T.T.G.A.A.mG.mU.mC.mC.mC-3’

ii. NTC ASO sequence: 5’-mC.mC.mU.mU.mC.C.C.T.G.A.A.G.G.T.T.mC.mC.mU.mC.mC-3’

2. Dissolve powdered ASO in buffer (10 mM Tris with 0.1 mM EDTA) to 100 μM

3. Aliquot the ASOs and store at -20^oC until use

a .We used ~ 50 μL aliquots

Apply Tau ASO 1 week before experimental timepoint:  

1. Thaw aliquot of ASO over ice

2. Apply ASO directly to neuronal media to a final concentration of 1 μM

a. E.g. for neurons in a 24-well plate with 500 μL media, add 5 μL of 100 μM aliquot to each well

b. Include wells with Tau ASO and Nontargeting control ASO in the same plate, and compare between wells within the same plate

3. Very gently, tip plate side-to-side to mix media without disrupting neurons or coverslips

4. 1 week after application, complete desired experiments

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