Protocols
Bench protocols for antisense oligonucleotides
Step by step, for the self-delivering and transfection-optimized antisense oligonucleotides and for in vivo work, with the controls and validation every experiment needs.
In cells
AUMsilence
sdASO protocol - no transfection reagent
- 5-20 μM
- 24-72 hours
Plate the cells, add the oligo directly to the culture medium, then read mRNA by qRT-PCR, protein by Western blot, ELISA or immunofluorescence, or a functional assay.
AUMantagomir
sdASO protocol - no transfection reagent
- 5-20 μM
- target de-repression
Plate, add to the medium, then confirm inhibition by the de-repression of known miRNA target genes, the more reliable measure, since a hybridized oligo may interfere with miRNA detection.
AUMlnc
sdASO protocol - no transfection reagent
- nuclear-retained lncRNAs
- 48-96 hours
Knockdown of long non-coding RNAs, including nuclear-retained transcripts inaccessible to traditional RNAi; start at 10 μM and allow 48-96 hours for nuclear targets.
AUMblock
sdASO protocol - no transfection reagent
- stoichiometric, not catalytic
- 10 μM to start
Block translation, a splice site, a microRNA binding site or a protein binding site without recruiting RNase H, and read the function rather than the RNA level.
AUMsplice
sdASO protocol - no transfection reagent
- primers flanking the exon
- 5-20 μM
Skip an exon by blocking its splice sites or exonic splicing enhancers; the skipped and non-skipped variants are read by RT-PCR and gel, and the altered protein by Western blot at 48-96 hours after treatment.
AUMsilence V+
sdASO protocol - no transfection reagent
- before or after infection
- MOI 0.01-0.1 to start
Target viral RNA in cells treated before or after infection, then read viral RNA by qRT-PCR, viral protein, infectious titer or cytopathic effect at 24-72 hours after treatment.
AUMsilence
toASO protocol - standard transfection methods
- 50-100 nM in the well
- 50-70% confluency
Delivered with a lipid, electroporation, calcium phosphate or polymer method, each with its own steps and the reagent volumes per plate format; knockdown is read at 24-72 hours after transfection.
AUMantagomir
toASO protocol - standard transfection methods
- 50-100 nM in the well
- target de-repression
Delivered with a lipid, electroporation, calcium phosphate or polymer method, with the reagent volumes per plate format; inhibition is confirmed by the de-repression of known miRNA target genes.
AUMlnc
toASO protocol - standard transfection methods
- 50-100 nM in the well
- nuclear-retained lncRNAs
Delivered with a lipid, electroporation, calcium phosphate or polymer method, with the reagent volumes per plate format; knockdown is read by qRT-PCR or RNA-FISH, 48-96 hours for nuclear targets.
In animals
sdASO in vivo protocol
- IV, SC, IP or IT
- 3-30 mg/kg in mice
- more than one timepoint
Formulate in PBS or saline, dose by route and body weight, then collect tissues at more than one timepoint and read knockdown by qRT-PCR, Western blot or ELISA.
Every experiment
Controls and validation strategies
- untreated and non-targeting controls
- dose response and time course
- RT-qPCR and Western blot
Which negative, positive and rescue controls every experiment needs, the dose response and time course, how knockdown is validated at the mRNA, protein and functional level, and how it is reported.
For research use only. Not for use in diagnostic or therapeutic procedures.