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Protocols

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FANA Antisense Oligonucleotides (FANA ASOs) are uniquely designed and manufactured using state-of-the-art chemical modifications that allow for highly efficient cell delivery in the absence of transfection reagents, thus eliminating cell toxicity associated with lipid transfection reagents and electroporation. FANA ASOs have effective self-delivery and superior performance and work in a wide range of cell types, difficult-to-transfect primary cells (B-cells, T-cells, neurons, etc.), as well as in vivo study models.

In vivo -ready FANA ASO is engineered to be simple and effective for self-delivery into animals without the need of special formulations or conjugations. They are highly purified for direct delivery. Self-delivering FANA ASOs can be eciently taken up by the target tissues, and can significantly increase theirstability and their resistance to endonucleases.

RNA Silencing Protocol

01

Seed cells in the desired format the day before treatment

Plate cells the day before (for adherent cells) or prior to treatment with FANAs (for suspension cells) in complete media at a 30% - 50% cell density (or at densities optimized for growth conditions and the end-point of the assay). In case of adherent cells, allow the cells to adhere.

02

Reconstitute lyophilized FANA ASOs in the appropriate amount of water or buffer . If the stock solution has already been prepared, skip to step 3.

Resuspend lyophilized FANA ASOs using the appropriate volume of sterile water or buffer. Pipette solution up and down 3-5 times while avoiding introduction of bubbles. Let the vial sit at room temperature for 5-10 minutes. Centrifuge for 30-45 seconds to collect solution to bottom of the tube. It is recommended to make several aliquots of the stock solution to avoid multiple freeze thaw cycles.

03

Add FANAs to the cells and media to the desired final concentration.

For silencing experiments, the working concentration of FANA ASOs can vary from 500 nM to 10 µM. It is highly recommended to perform a dose response using 2-3 working concentrations (500 nM, 5 µM and 10 µM) to determine the most optimum concentration for your application. In some specific cases, 20 µM or higher concentrations may be required. For adherent cells, aspirate the growth media and overlay cells with media containing FANAs or add FANAs stock directly to the media overlaying the cells. Mix gently. For suspension cells, pellet the cells by low-speed centrifugation and gently resuspend the cell pellet in media containing FANAs. Alternatively, add FANAs ASOs directly to the media overlaying the cells.

04

Analyze FANA treated cells after 24-72 hours post treatment.

Uptake of fluorescently-labeled FANA can be observed as early as 4-8 hours, but full knockdown is best assessed at 24-96 hours post treatment.

NOTES

The above is a general protocol for the use of FANA in mammalian cells. It can be adapted for different cell types and different culture vessel formats.

Cell Culture Plate 96-well 24-well 12-well 6-well
FANA ASO* stock (µL) 1 µL 5 µL 10 μL 30 μL
FANA ASO* used (moles) 100 pmole 500 pmole 1 nmole 3 nmole
silenceONE 100 µL 500 µL 1000 µL 3000 µL
Cell number (per well) ** 0.5x10 5 2.5x10 5 0.5x10 6 1.0x10 6

* The amount of FANA shown yields a final concentration of 1 µM using 100 µM stock

** The optimal seeding cell density will vary with the cell type, cell size, growth characteristics and the end-point of the assay. For this table, HeLa cells at 50% confluency were used

at the time of FANA treatment. In general, a confluency of 30 – 50% is recommended at the time of FANA ASO treatment.

FANA ASO Calculation Guidelines
NOTES

Depending upon the experiment, different time points can be used to measure knockdown or related effects for up to several days (and weeks in some cases) using a single dose.

In certain cases (especially for very fast-growing cells), the knockdown effect may be reduced after a few days. In such cases, simply add more FANA ASOs to the cell culture to maintain knockdown.

FANA ASOs can be fluorescently-labeled (or with any desired label) to monitor cellular uptake.

Storage

01

FANA ASOs are shipped in lyophilized form. Upon arrival, store them in -20°C.

02

When ready to use, resuspend FANA ASOs in sterile water or your favorite buffer at the desired concentration. Aliquot resuspended FANAs in working aliquots to avoid multiple freeze-thaw cycles.

01

Prepare a FANA ASO stock solution by reconstituting lyophilized FANA ASOs at the desired concentration.

Resuspend FANA ASOs using the appropriate volume of sterile water or saline buffer. We recommend a stock solution of 5-10 mg/ml. The approximate molecular weight of a 21 nt single-stranded FANA ASO is between 7000-8500 g/mol (sequence-dependent). Pipette solution up and down 3-5 times while avoiding introduction of bubbles. Let the vial sit at room temperature for 5-10 min. Briefly centrifuge to collect solution to bottom of the tube. Then, calculate FANA concentrations for in vivo delivery.

For in vivo systemic delivery, we recommend a dose between 3-30 mg/kg in mice. If topical (organ, intratumoral, etc.) delivery is desired, the concentration can be adjusted accordingly considering the volume of the organ or tumor. We recommend you perform a dose response using 2-3 working concentrations to determine the most optimum concentration for your specific application. If long-term gene silencing is desired, multiple doses of FANA can be administered.

02

Administer reconstituted FANA ASOs to animal via the preferred route.

FANA oligos can be administered via different routes: intravenous (IV), intraperitoneal (IP), intradermal (ID), intratumoral, intranasal, intratracheal, hydrodynamic tail injection, inhalation, or local organ delivery.

03

Assay for gene knockdown in your target tissues.

In most cases, uptake of fluorescently-labeled FANA can be observed as early as 24-72 hours, but in vivo knockdown can be assessed at 72 hours and beyond post FANA administration.

NOTES

The above is a general protocol for the use of FANA in mouse models. It can be adapted for use in other mammalian or in vivo models.

NOTES

Depending upon the experiment, different time points can be used to measure knockdown or related effects for up to several days (weeks and months in some cases) using a single dose.

If prolonged knockdown is desired, FANA ASOs can be re-administered every few days/weeks.

FANA ASOs can be fluorescently-labeled (or with any desired label) to monitor cellular uptake or bio-distribution.

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