7 Tips for Optimal DNA Transfection

DNA transfection is a main tool for current genetics as well as cell and molecular biology studies. Understanding the underlying mechanisms and the different parameters affecting transfection is crucial for optimal, reproducible, and trustable results. Following these seven tips will ensure reliable results.

1. Adapt the transfection conditions to your experiment. Transfection conditions should be optimized according to the cells, the size of the plasmid, the number of plasmids, the expression pattern of the gene of interest, the culture vessel, and the transfection reagent. Reoptimize the conditions for each new cell line.
2. Transfect healthy cells. Passage cells at least twice after thawing to allow recovery before transfection, and use cells at low passage number (< 20 passages). Discard overconfluent cells. Regularly check for mycoplasma contaminations. Seed cells the day before transfection accordingly to the confluency recommended by the transfection reagent provider.
3. Follow the transfection reagent protocol. Some reagents are inhibited by serum or antibiotics, while others may be used in serum- and antibiotic-containing medium, hence reducing the risks of toxicity and the number of steps in the protocol. Check the recommended cell confluency, as well as the recommended DNA amount and reagent volume.
4. Work with high-quality DNA preparation. Check for RNA contamination by agarose gel electrophoresis and ethidium bromide staining. Measure UV absorbance at 280 nm. OD260/280 ratio should reach at least 1.8. Resuspend the plasmid in deionized water or TE buffer at a concentration of ca. 1 µg/µl. Aliquot the plasmid preparation and store at -20°C to avoid freeze/thaw cycles.
5. Check serum quality. Some serum lots may inhibit drastically transfection efficiency, hence resulting in lower silencing efficiency. Check transfection efficiency of different serum lots before purchasing a new batch of serum. Also ensure that the medium used allows for efficient transfection, as some media coumpound may decrease transfection efficiency.
6. Minimize cytotoxicity by using low DNA amount and low transfection reagent volume. Check that the target gene does not affect cell viability. Analyze transfection at an earlier time point (24 h after transfection instead of 48 h for instance).
7. Use appropriate controls. Use a reporter gene to set up and optimize transfection conditions, as those may vary depending on the cells to transfect. Various reporter systems are commercially available: Renilla Luciferase and GFP (Green Fluorescent Protein) are the most commonly used.

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Desmoid tumor: benign but nasty

Desmoid tumor, also called deep-seated fibromatosis, is a benign tumor that is nonetheless a nasty beast. It is composed of fibroblasts, and it often presents as a large, infiltrative masses. Desmoid tumors may occur in patients with familial adenomatous polyposis (also called Gardner syndrome), an autosomal dominant disorder characterized by innumerable colon polyps (hence the name), as well as other lesions such as osteomas, jaw osteomas, odontomas, thyroid carcinoma, epidermoid cysts, fibromas, and sebaceous cysts. If the colon is not removed in a patient with familial adenomatous polyposis, the chance of colon carcinoma is virtually 100%.

Back to desmoid tumors. These tumors usually present as firm, rubbery, ill-defined, infiltrative masses. They may occur in extra-abdominal regions (for example, in the shoulder or thigh), within the abdominal wall itself (often attached to the rectus abdominus), or in the intra-abdominal region. Histologically, desmoid tumors are composed of bland-appearing fibroblasts in broad fascicles that infiltrate surrounding tissue. Check out the photo above: you can see muscle fibers being splayed apart by benign-looking, round-to-spindly tumor cells.

Although benign, desmoid tumors can act nasty. They are locally aggressive and invasive, and if you don't excise them completely, they are likely to recur. Treatment with things that you normally only use for malignancies, such as tamoxifen, chemotherapy and radiation, has been effective in many cases.

Mastocytosis

Mastocytosis is actually a spectrum of rare disorders, all of which are characterized by - not surprisingly - an increase in mast cells. Most patients have disease that is localized to the skin, but about 10% of patients have systemic involvement. There is a localized, cutaneous form of mastocytosis called urticaria pigmentosum that happens mostly in children and accounts for over half of all cases of mastocytosis.

Clinically, the skin lesions of mastocytosis vary in appearance. In urticaria pigmentosum, the lesions are small, round, red-brown plaques and papules. Other cases of mastocytosis show solitary pink-tan nodules that may be itchy or show blister formation. The itchiness is due to the release of mast cell granules (which contain histamine and other vasoactive substances).

In systemic mastocytosis, patients have skin lesions similar to those of urticaria pigmentosum - but there is also mast cell infiltration of the bone marrow, lymph nodes, spleen and liver. Patients often suffer itchiness and flushing triggered by certain foods, temperature changes, alcohol and certain drugs (like aspirin).

Histologically, mast cell infiltration may be subtle (with ocasional spindle-shaped mast cells around vessels in the superficial dermis) to dramatic (with tightly-packed mast cells in the upper to mid-dermis - check out the photo above). Mast cells may be difficult to distinguish in regular H&E sections; you may need a metachromatic stain (toluidine blue or Giemsa) to really visualize the granules.

In Virto Fertilization IVF Third Edition - Brian Dale, Kay Elder

Review

Review of the previous edition: '... an excellent handbook for good IVF laboratory practice right down to a description of total quality management (TQM) systems ... concise, well written and should be mandatory reading for all potential and actual embryologists, as well as for those clinicians seeking a wider understanding of the subject.' Human Fertility

Review of the previous edition: 'The authors are masters of this work. Their views are up-to-date, practical, and well documented. This work will be of greatest interest to clinical practitioners of in vitro fertilization that want a relatively short and very precise overview of laboratory methods. It is highly recommended.' John E. Buster, Fertility and Sterility

Book Description

Fully updated with new practical techniques, this new edition describes the form and function of human gametes and embryos, and how this information is used in modern IVF practice. Packed with a wealth of practical and scientific detail, this popular guide is a must for all IVF students and practitioners.

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