Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.

Fournier's gangrene (FG) is a rare but life threatening disease. Although originally thought to be an idiopathic process, FG has been shown to have a predilection for patients with diabetes as well as long term alcohol misuse; however, it can also affect patients with non-obvious immune compromise. The nidus is usually located in the genitourinary tract, lower gastrointestinal tract, or skin. FG is a mixed infection caused by both aerobic and anaerobic bacterial flora. The development and progression of the gangrene is often fulminating and can rapidly cause multiple organ failure and death. Because of potential complications, it is important to diagnose the disease process as early as possible Although antibiotics and aggressive debridement have been broadly accepted as the standard treatment, the death rate remains high.

Priapism is a pathological condition of penile erection that persists beyond, or is unrelated to, sexual stimulation. Pathologically and clinically, two subtypes are seen—the high flow (non-ischaemic) variety and the low flow (ischaemic) priapism. The low flow type is more dangerous, as these patients are susceptible to greater complications and the long term recovery of erectile function is dependent on prompt and urgent intervention. Many of the causes of priapism are medical, including pharmacological agents, and as such, priapism should be considered as a medical and surgical emergency.

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This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in ; Vol. 22, No. 39. See the NIHR Journals Library website for further project information. This project was also supported and partially funded by the NIHR Biomedical Research Centre at University College London (UCL) Hospitals NHS Foundation Trust and UCL and by The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research Biomedical Research Centre and was co-ordinated by the Medical Research Council's Clinical Trials Unit at UCL (grant code MC_UU_12023/28). It was sponsored by UCL. Funding for the additional collection of blood and urine samples for translational research was provided by Prostate Cancer UK.

Background All risk stratification strategies in cancer overlook a spectrum of disease. The Prostate MR Imaging Study (PROMIS) provides a unique opportunity to explore cancers that are overlooked by multiparametric magnetic resonance imaging (mpMRI). Objective To summarise attributes of cancers that are systematically overlooked by mpMRI. Design, setting, and participants PROMIS tested performance of mpMRI and transrectal ultrasonography (TRUS)-guided biopsy, using 5 mm template mapping (TPM) biopsy as the reference standard. Outcome measurements and statistical analysis Outcomes were overall and maximum Gleason scores, maximum cancer core length (MCCL), and prostate-specific antigen density (PSAD). Cancer attributes were compared between cancers that were overlooked and those that were detected. Results and limitations Of men with cancer, 7% (17/230; 95% confidence interval [CI] 4.4–12%) had significant disease overlooked by mpMRI according to definition 1 (Gleason ≥ 4 + 3 of any length or MCCL ≥ 6 mm of any grade) and 13% (44/331; 95% CI 9.8–17%) according to definition 2 (Gleason ≥ 3 + 4 of any length or MCCL ≥ 4 mm). In comparison, TRUS-guided biopsy overlooked 52% (119/230; 95% CI 45–58%) of significant disease by definition 1 and 40% (132/331; 95% CI 35–45%) by definition 2. Prostate cancers undetected by mpMRI had significantly lower overall and maximum Gleason scores ( p = 0.0007; p < 0.0001) and shorter MCCL (median difference: 3 mm [5 vs 8 mm], p < 0.0001; 95% CI 1–3) than cancers that were detected. No tumours with overall Gleason score > 3 + 4 (Gleason Grade Groups 3–5; 95% CI 0–6.4%) or maximum Gleason score > 4 + 3 (Gleason Grade Groups 4–5; 95% CI 0–8.0%) on TPM biopsy were undetected by mpMRI. Application of a PSAD threshold of 0.15 reduced the proportion of men with undetected cancer to 5% (12/230; 95% CI 2.7–8.9%) for definition 1 and 9% (30/331; 95% CI 6.2–13%) for definition 2. Application of a PSAD threshold of 0.10 reduced the proportion of men with undetected disease to 3% (6/230; 95% CI 1.0–5.6%) for definition 1 cancer and to 3% (11/331; 95% CI 1.7–5.9%) for definition 2 cancer. Limitations were post hoc analysis and uncertain significance of undetected lesions. Conclusions Overall, a small proportion of cancers are overlooked by mpMRI, with estimates ranging from 4.4% (lower boundary of 95% CI for definition 1) to 17% (upper boundary of 95% CI for definition 2). Prostate cancers undetected by mpMRI are of lower grade and shorter length than cancers that are detected. Patient summary Prostate cancers that are undetected by magnetic resonance imaging (MRI) are smaller and less aggressive than those that are detected, and none of the most aggressive cancers are overlooked by MRI.

Recent research in molecular biology has identified a significant number of novel markers, which may have diagnostic, prognostic and therapeutic significance. This is particularly pertinent in the field of cancer. Validation of these markers in multiple clinical specimens is currently performed by traditional histopathological techniques, which are disappointingly time consuming, labour intensive and, therefore, economically costly. These limitations have hampered the introduction of many novel markers into everyday clinical practice. The tissue microarray (TMA) is a high throughput technique, which allows the rapid and cost effective validation of novel markers in multiple pathological tissue specimens. Tissue from up to a 1000 histology blocks can be arrayed accurately onto a newly created paraffin block, at designated locations. Subsequently, morphological and molecular investigations can be performed to determine the clinical significance of the novel markers tested. It is now firmly established that the TMA can significantly accelerate the processing of a very large number of tissue specimens with excellent quality, good reliability and preservation of original tissue, with ultimate clinical benefit.

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