A simple, cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection.

Understanding the molecular basis of disease requires gene expression profiling of normal and pathological tissue. Although the advent of laser microdissection (LMD) has greatly facilitated the procurement of specific cell populations, often only small amounts of low quality RNA is recovered. This precludes the use of global approaches of gene expression profiling which require sizable amounts of high quality RNA. Here we report a method for processing of snap-frozen tissue to prepare large amounts of intact RNA using LMD. Portions of small intestine from piglets (n = 6) were snap-frozen in Optimum Cutting Temperature compound (experimental) and in RNAlater (control). A randomly selected sample was laser microdissected using the developed protocol in multiple sessions totalling 4 h each day on four consecutive days. RNAs were extracted from these samples and its control and their quality (RIN) determined. RINs of the experimental samples were independent of time (p = 0.12) and day (p = 0.56) of the microdissection thereby suggesting that their RNA quality remained unaltered. These samples exhibited high quality (RIN ≥ 8) with good recovery (81.2%) and excellent yield (1539 ng/1.2 × 10(7) µm(2)). Their overall RIN, 8.029 ± 0.116, was not significantly different from 8.2 (p = 0.123), the value obtained from the control, non-laser microdissected, sample. This indicated that the RNA quality from the laser microdissected and non-microdissected samples was comparable. The method allowed LMD for up to 4 h each day for a total of four days. The microdissected samples can be pooled thereby increasing amount of RNA at least by ten-fold. The procedure did not require any expensive limited-shelf life RNase inhibitors, RNA protectors, staining kits or toxic chemicals. Furthermore, it was flexible and enabled the processing without affecting routine laboratory workflow. The method developed was simple, inexpensive and provided substantial amounts of high quality RNA suitable for gene expression profiling and other cellular and molecular analyses for biology and molecular medicine.

Isolation of guard cells from fresh epidermis using a piezo-power micro-dissection system with vibration-attenuated needles.

The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.

An ultrasonically actuated silicon-microprobe-based testicular tubule assay.

Microdissection testicular sperm extraction (TESE) is an invasive surgical procedure in which sparsely located healthy larger diameter tubules carrying viable spermatazoa are identified by visual examination of the seminiferous tubules of the infertile testis under a microscope, and biopsies of regions of interest are performed. In this paper, we report on microfabricated silicon microprobes integrated with an ultrasonic horn actuator and strain gauges for microdissection probe-TESE (MP-TESE) surgery. The microprobes, with axial-force-sensitive polysilicon strain gauges, have high force sensitivity (-0.4 V/N). The probes were used to detect the boundaries between seminiferous tubules, thus enabling identification of individual tubule diameters. Insertion experiments were performed on rat testis tissue, and by monitoring the tubule puncture in the recorded force, we were able to estimate the average diameter approximately 41.2 +/- 1.6 microm of the sperm-carrying tubules in samples. We have also demonstrated the ability to sense the existence of larger tubules embedded in a mess of thinner tubules, using an analytically calculated expression for the distribution of sizes measured by the microprobe. This information is important in MP-TESE to distinguish tubules with and without fertile sperm, potentially eliminating the large incision currently required for optical spermatazoa localization.

Preparation and quality assessment of RNA from cell-specific samples obtained by laser microdissection.

Laser microdissection is a powerful tool to obtain cell-specific isolates from complex tissue samples. This chapter outlines how to prepare plant material for microdissection and methods to extract and measure high-quality RNA suitable for a variety of different downstream applications.

Influence of new technologies on thyroid surgery: state of the art.

The history of thyroid surgery starts with Billroth, Kocher and Halsted, who developed the technique for thyroidectomy between 1873 and 1910. In general, the essential objectives for thyroidectomy are conservation of the parathyroid glands, avoidance of injury to the recurrent laryngeal nerve, an accurate hemostasis and an excellent cosmesis. In the last 20 years, major improvements and new technologies have been proposed and applied in thyroid surgery; among these are mini-invasive thyroidectomy, new devices for achieving hemostasis and dissection, regional anesthesia and intraoperative neuro-monitoring.

Combining ultrasonic dissection and the Storz operation rectoscope.

BACKGROUND: Transanal endoscopic microsurgery (TEM) allows a precise, full-thickness resection of rectal tumors anywhere within the rectum. Unfortunately, the standard TEM technique needs complex and rather expensive equipment, demands high skill, and is attended by bleeding and oozing that may be challenging. A modified TEM procedure combining the new Storz operation rectoscope and ultrasonic dissection has been developed to overcome the limitations of the original technique. METHODS: The Storz operation rectoscope features a 5-mm telescope combined with a single-monitor display. Standard laparoscopic instruments and the LCSC5 Ultracision Maniple are used for dissection and coagulation. Full-thickness resection is performed most often. Closure of the defect is accomplished by interrupted 3-0 polydoxanone sutures secured by extracorporeal slipknots. RESULTS: Altogether, 18 TEMs have been performed according to the modified technique: 9 for malignant and 9 for benign lesions. The median operating time was 92.5 min for resection of malignant lesions and 40 min for resection of benign lesions. Two postoperative complications occurred: a bleeding and a partial dehiscence. The median follow-up periods were 35 months for malignant disease and 19.5 months for benign disease. No recurrence was observed. CONCLUSION: For tumors located up to 15 cm from the anal verge, TEM with the Storz rectoscope and ultrasonic dissection is indicated. Despite the complication described, coagulation is optimal and ultrasonic scissors allow working in a fairly bloodless field. The overall costs of the equipment are significantly lower.