Introduction by microinjection can be used for any type of adherent cell, including primary cells. Since siRNA for any target gene can be obtained simply and rapidly, it is a relatively simple method to study multiple effects of any one gene knockdown in any kind of adherent cell within a few days. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Microinjection of siRNA into mammalian cells Move Microinjection of siRNA into mammalian cells Materials reagents Cell lines injected (e.g., adherent cell lines used here) Fluorescently labeled dextran (molecular probe/In vitrogen ) We recommend the use of dextran that has been chemically modified with an amino group that allows dextran to be immobilized by acetaldehyde. Dextran with a molecular mass around TOkDa is also recommended to avoid crossing the nuclear membrane. Oligonucleotides siR N A siRNAs and control s;r n a for the target gene can be designed using the online software provided by the supplier. The oligonucleotide was denatured and prepared as a 20umol/L storage solution and stored at 20°C. The oligonucleotide was then used as a storage solution. siRNA Sample Buffer 5 mmol/L Pityrosine (p H 7.2) 100 mmol/L K C l Instrumentation Coverslips (acid-washed) Drop-shot fluorescence microscopy for analyzing most experimental results High-quality inverted optical microscope with multiple objective lenses Semi-automatic microinjection system with adjustable injection voltage and time as well as micro manipulator and lever. Microinjection equipment is available from several manufacturers and has been described in detail elsewhere (R oseet al.1999; see Ikeda2004 for a detailed description; King 2004). Microinjection needles are available for purchase; we have successfully manufactured our own commercially available needle removal tool. Methods. 1. Prepare a working solution of s i R N A oligonucleotides (specific and control oligonucleotides). s i R N A oligonucleotides were prepared at a concentration of 50n m o l/L in s i R N A sample buffer. Dissolve fluorescently labeled dextran at 5m g /m l in s i R N A sample buffer. 2 . To prepare s i R N A for microinjection, dilute the working solution of s i R N A oligonucleotides 1 0-fold into the labeled dextran solution. This picomolar concentration is effective and sufficient to avoid off-target nonspecific effects. 3 . Spread the adherent cells on acid-washed coverslips and grow to a suitable density of about 5 0 % confluence. 4 . Microinjection of the nuclei is performed according to standard procedures using prepared specific or control s i R N A samples (see, e.g., King2004 and Fig. 1). DNA, peptides, and other living molecules suitable for the study system can also be used (ZhuetaL 2006). 5. After microinjection, the cells are incubated for a period of time to allow sufficient knockdown of the target protein. 6 . The analysis is applicable to cells in the study system. An important aspect of the methodology is the analytical approach used to evaluate microinjections. The phenotypes generated by siR N A must be calibrated using an objective indicator, especially one that can classify single cells per injection as positive or negative. Good samples can be constructed using bromodeoxyuridine doping (Lie t al. 2002), immunocytochemical staining with appropriate markers (Klappacher et al. 2002), or using co-injected promoter/reporter gene constructs (p erissie ta l. 2004; Zhu et al. 2006). For more product details, please visit Aladdin Scientific website.
At that point, serum may be removed if necessary to quench the cells.
The siRN A effect is rapid and specific. In most cases, changes in the target inRNA are not detectable by RT-PCR within 6 h of injection. The time period of the process varies with the half-life of the target protein, but most regulatory proteins have relatively short half-lives.