The steps of mRNA transfection


Posted January 4, 2022 by Bonnibelle

BOC Sciences is a life science group with its headquarter in New York. We are dedicated to providing the most complete in vivo and in vitro nucleic acid and protein transfection

 
mRNA transfection steps (24-well plate)
1. Cell planting 1 day in advance
Adherent cells: Plant the cells in a 24-well plate one day in advance. It is advisable that the cell confluence is about 30% during transfection. The total amount of the whole medium before transfection is 0.45ml.
Suspension cells: use cells in logarithmic growth phase, the number of which is 1/3 of the number of conventional cultured cells for transfection experiments. If the number of cells in a regular culture is 6×105, then use 2×105 cells for transfection.
2. Transfection process
⑴ Take 1ug (50pmol) of mRNA, add a certain amount of serum-free diluent, mix well to make RNA diluent, the final volume is 25μl.
Note: It is recommended to use OPTI-MEM, serum-free DMEM or 1640 as the serum-free diluent.
⑵Take 1.5ul of Entranster-R4000, then add 24ul of serum-free dilution liquid, mix well to make Entranster-R4000 dilution, the final volume is 25μl. Let stand at room temperature for 5 minutes.
(3) Mix Entranster-R4000 diluent and RNA diluent thoroughly (vibrate with a shaker or pipette more than 10 times), mix, and let stand at room temperature for 15 minutes. The preparation of the transfection complex is complete.
⑷ Add 50μl of transfection complex dropwise to the cells with 0.45ml of complete medium (10% serum and antibiotics can be contained), move the petri dish back and forth, and mix well.
Note: For this reagent, the use of serum-containing complete medium will help improve transfection efficiency.
⑸ Observe the cell status 6 hours after transfection. If the condition is good, there is no need to change the medium. Continue to culture for 24-96 hours to get the result.

How to choose a DNA transfection reagent

Obviously, the key to the success of plasmid transfection lies in the choice of plasmid transfection reagents. At present, the commonly used DNA transfection reagents include RFect series plasmid DNA transfection reagents, Lipo2000 and so on. How to choose a suitable plasmid DNA transfection reagent can generally be considered from the following aspects:

1. High transfection efficiency for plasmid DNA. To determine the transfection efficiency, methods such as fluorescence quantitative PCR, fluorescence microscopy, and flow cytometry are commonly used. The gold standard is undoubtedly the detection of the mRNA level of the target gene, because the transfection reagent not only needs to transfect the plasmid DNA into the cell, but also release the DNA transferred into the cell in time to play the role of gene expression or gene regulation. There are at least two problems in judging the transfection efficiency by fluorescence microscopy: 1. The fluorescent spots seen by the fluorescence microscope may be the result of non-specific adsorption; 2. Some transfection reagents can transfect plasmid DNA into cells, but they cannot be fully released. The efficiency of gene expression or gene regulation is not high. Therefore, the gold standard for judging the transfection efficiency of transfection reagents should be the fluorescence quantitative detection of mRNA level. Only observing the fluorescence microscope to judge the transfection efficiency and choosing the transfection reagent will often lead to misjudgment and affect the progress of the experiment. As far as transfection efficiency is concerned, the transfection efficiency of RFect Plasmid DNA Transfection Reagent for most adherent cells can be as high as 90% or more (EGFP plasmid). In addition, RFectSP Suspension Cell Plasmid DNA Transfection Reagent is specifically for plasmids for suspension cells. DNA transfection, the transfection efficiency is as high as 70% in most suspension cells. In contrast, the transfection efficiency of Lipo2000 for most adherent cells is generally about 75%, and it is impossible to effectively transfect some primary cells and suspension cells. Obviously, in terms of transfection efficiency, the RFect series of plasmid DNA transfection reagents have more advantages.
2. Very little toxicity to transfected cells. If the transfection reagent is too toxic, it will cause a large number of cell deaths, which will directly affect the interpretation and interpretation of the results of the transfection test. Liposomal transfection reagents themselves can cause changes in the expression of certain genes, thus affecting the objectivity of the transfection test results. It can be seen from the above that transfection reagents with less toxicity should be selected as far as possible.
3. Types of transfected cells. Lipo2000 is unable to effectively transfect primary and suspension cells, and because primary cells are more sensitive than domesticated cell lines, the mortality rate after Lipo2000 transfection is also high. RFect series plasmid DNA transfection reagents have low cytotoxicity. Depending on the types of cells to be transfected, you can purchase RFect plasmid DNA transfection reagents or RFectSP suspension cell plasmid DNA transfection reagents.

About Us
BOC Sciences is a life science group with its headquarter in New York. We are dedicated to providing the most complete in vivo and in vitro nucleic acid and protein transfection solutions to support gene and cell therapy, biologics production, and life science research. Here are some our products: dna transfection, viral transfection, cell transfection, lipotransfection, etc.
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Issued By www.bocsci.com
Country United States
Categories Biotech
Last Updated January 4, 2022