Tips for DNA Transfection
Cells
Do not use freshly thawed cells. After thawing and before transfection, passage your cells at least 3 times, in order to give them time to recover and return to their normal behavior. Passage your cells when they are in log-phase growth and before they reach confluency. Plate your cells the day before transfection in order to achieve 60-80% of visual confluency on the day of transfection.
Cells should be healthy and actively dividing, they should not be in culture for too long (> 8 weeks). Cell viability can be assessed using OZ Biosciences' WST-8 Cell Proliferation Kit or MTT Kit (#MT01000) and senescence can be checked with OZ Biosciences'senescence kit (#GXS003).
Cells and media must be free of contamination (mycoplasma, yeast, bacteria). Cells should be maintained in optimal culture conditions.
DNA Quality
DNA solution must be prepared in DNase/RNase free water or TE buffer. DNA must be as pure as possible, free of contaminants (proteins, lipids, carbohydrates) and endotoxins.
Abs 260/280nm of the DNA solution should be between 1.7 and 1.9. Do not use DNA solution with lower or higher ratio for transfection. Ensure that your promoter is compatible with the cells to be transfected.
Ratio of Transfection Reagent
It's critical to test multiple reagent:DNA ratios to find the right ratio that achieves the highest transfection efficiency with minimal toxicity. Optimize the reagent/DNA ratio by using a fixed amount of DNA (µg) and varying the volume of the transfection reagent. For example, vary the concentration of Helix-IN Transfection Reagent from 1–2.5 μl per 1 μg DNA to find the optimal ratio.
DNA Amount
It is important to refer to the specific protocol of the reagent you are using and to keep in mind that all cells don’t require the same quantity of DNA.
Use different quantities of DNA as suggested in the instruction manual/related protocol with the optimized tranfection reagent:DNA ratio. You may adjust the amount of DNA depending on the transfected cell lines and the transfection reagent.
Be sure to keep DNA:Reagent ratio constant when adjusting DNA dose.
Controls
Positive controls are highly recommended for your transfection experiments. OZ Biosciences offers a large range of pVectOZ control plasmids encoding the most common reporter genes (GFP, b-Gal, Luciferase, SEAP, CAT).
Mock transfection: perform a transfection control without any DNA so that eventual non-specific effects due to the transfection reagent can be observed.
Transfection Practices
We recommend preparing the DNA/reagent complexes in DMEM or PBS without any supplement.
Allow the reagent to reach room temperature before starting your experiment.
Once DNA is added to the transfection reagent, incubate for 20 min at room temperature for optimal complexes formationand add directly the mix onto your cells.
Do not wait for more than 30 minutes once the DNA/reagent complexes are formed.
When using Helix-IN do not incubate complexes Helix-IN/DNA less than 30 min at RT
Distribute the complexes onto the cells in a dropwise manner and gently rock the plate to ensure even dispersion.
N/P Charge Ratios
Numerous research studies reported the importance of the charge ratio between the positive cationic lipids and the negative DNA to generate lipoplexes for successful plasmid delivery. These assessments concluded to a considerable effect of the charge ratio on physicochemical features of the lipoplexes and subsequently on the transfection efficiency. Usually, increasing the charge ratio leads to improving electrostatic interaction between the cationic lipid and the DNA. Nevertheless, above the optimal charge ratio it has been found that a dose-dependent increase of cytotoxicity occurs. This charge ratio is known at the N/P ratio, calculated based on the positive charge from the amino groups in the cationic lipids (N) and the phosphate group of the DNA backbone (P) (or the negative charges of the amino acids in proteins) concentration. Herein, DOTAP has one nitrogen carrying one positive charge. As a direct consequence, 1 nmol of DOTAP contribute to 1 nmol of positive charge. On the other hand, 1 µg of DNA contributes to 3.1 nmol of negatively charged phosphate.[1] Mohammed-Saeid, W., et al. Di-peptide-modified gemini surfactants as gene delivery vectors: exploring the role of the alkyl tail in their physicochemical behavior and biological activity. The AAPS journal, 2016, 18, 1168-1181. [2] Datta, D., et al. Impact of the Charge Ratio on the In Vivo Immunogenicity of Lipoplexes. Pharmaceutical Research, 2017, 34, 1796-1804.
Tips for siRNA Transfection
Cells
Do not use freshly thawed cells. After thawing and before transfection, passage your cells at least 3 times in order to give them time to recover and return to their normal behavior.
Passage your cells when they are in log-phase growth and before they reach confluency.
Plate your cells the day before transfection in order to reach 60-80% of visual confluency on the day of transfection.
Cells should be healthy and actively dividing, they should not be in culture for too long (> 8 weeks). Cell viability can be assessed using OZ Biosciences' MTT Kit (#MT01000) and senescence can be checked with OZ Biosciences' senescence kit (#GXS003).
Cells and media must be free of contamination (mycoplasma, yeast, bacteria).
Cells should be maintained in optimal culture conditions.
siRN
Use RNase-free materials.
Use high quality siRNA (PAGE purified and desalted).
If possible, work with low siRNA concentrations (1-10nM) in order to avoid off-target and/or cytotoxic effects.
Do not use water to diulte your siRNA stock solution, prefer the manufacturer's buffer or 100mM NaCl in 50mM Tris (pH 7.5) to avoid denaturation.
Calculate the correct amount of siRNA: the molecular weight of an average siRNA molecule is approx. 13.500.
Controls
Positive control: use siRNA directed against a housekeeping gene (i.e. GAPDH) or labeled siRNA.
Negative control: use mismatch and/or non-targeting sequence as negative control.
Transfection Practices
We recommend preparing the siRNA/reagent complexes in DMEM or PBS without any supplement. Allow the reagent to reach room temperature.
Once siRNA is added to the transfection reagent, incubate for 20 minutes at room temperature for optimal complexes formation and add directly the mis onto your cells. Do not wait for longer than 30 min once the complexes are formed.
Distribute the complexes onto the cells in a dropwise manner and gently rock the plate to ensure an even dispersion.
Optimal silencing after siRNA transfection can be obtained after 24h for mRNA expression assay up to 72h for protein expression assay.
Learn more about Oz Transfection Reagents here.