Summary of single-tube reactions.

<p>(A) Single-tube nucleic acid purification. All reagents (including lysis buffer containing PFC and wash buffer) can be added in one step, greatly simplifying the procedure. After a brief and intense vortex a homogenization state is achieved (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158018#sec012" target="_blank">discussion</a>). Nucleic acids are then purified by 1 μm, dextran-coated and positively charged magnetic beads. (B) Single-tube PCR. After isolation, reagents for use in nucleic acid amplification can be added directly into the same tube without further treatment, or, all the reagents required for nucleic acid isolation and amplification (including lysis buffer, washing buffer and PCR reagents) can be added in one step in the very beginning of the experiment. PCR is then conducted in PFC micelles. The amplified products are collected and purified by coated magnetic beads. (C) Single-tube emulsion PCR. After cell lysis and magnetic bead purification of nucleic acids, reagents for emulsion PCR can be added directly into the same tube after discarding the supernatant and the cellular debris. Tests can be performed in microemulsions (water-in-oil droplets) containing sequence-specific captures and probes for further high-throughput detection. The tube sizes in each column are not to scale.</p

cDNA yields and qualities of single-tube reaction samples.

<p>(A) PCR amplification of the <i>GAPDH</i> gene, performed in one tube and in one step with the nucleic acid isolation. cDNA yields and qualities of samples amplified using single-tube reactions were comparable to those amplified using commercialized kits (Fermentas Maxima^®) in two steps after nucleic acid isolation. (B) Standard dengue virus sera samples were tested by RT-PCR to see if trace amounts of specific sequences could be detected. Amplified products from single-tube reaction and commercialized kits were comparable.</p

Single-tube nucleic acid amplification using various sample volumes.

<p>Single-tube nucleic acid amplification using various sample volumes.</p

RNA isolation/purification from frozen mouse liver tissue, ITRI (I, duplicate), and Qiagen (Q) samples analyzed by Agilent 2100^® gel electrophoresis.

<p>Compared with the conventional methods, using single-tube reaction greatly simplified the procedures (see above). Analysis by Agilent 2100^® bioanalyzer revealed that the sample qualities were comparable. This demonstrates that by using single-tube reaction the experiment time can be greatly reduced while the nucleic acid quality is maintained.</p

Single-tube emulsion PCR.

<p>(A)(B)(C) Target 1 represents the DNA-probe-Cy3 in which the probe 1-Cy3 was labeled. Target 2 represents the DNA-probe-Cy5 in which the probe 2-Cy5 was labeled. WL represents the condition under white light. The results indicate that the biosample treated with a single-tube reaction was in complete oil-ball shapes after being amplified with emulsion PCR, which was beneficial for the next optical analyses. (D)(E) The formation of droplets is shown, which could be placed in arrays. The bright spots inside the droplets are fluorescent emissions from specifically labeled probes. N.C. represents negative control.</p

Overall flow chart comparing conventional and single-tube methods.

<p>We introduce the use of perfluorocarbon (PFC) for cell lysis and magnetic particles for nucleic acid purification in single-tube reaction. Compared with the conventional methods, using only one tube greatly simplify the procedures and reduce the risk of cross-contamination. No incubation or enzyme digesting time is needed.</p

Clinicopathological features and prognosis of patients with de novo versus nevus-associated melanoma in Taiwan

<div><p>Studies surveying melanomas associated with melanocytic nevi in Asia are rare. In this study, we examined whether nevus-associated melanomas differ from de novo melanomas in terms of their associations with clinical factors, histologic characteristics, and patient survival in Taiwan. Using data on cancer cases obtained from the Department of Pathology archives and the Cancer Registry of National Taiwan University Hospital, we conducted a retrospective analysis of 103 consecutive melanoma patients who were diagnosed between 2010 and 2015 and received follow-up through November 2016. Approximately 17.5% of the melanomas in question were associated with a nevus. In patients under 65 years of age, non-acral lentiginous melanomas were significantly associated with a higher percentage of nevus-associated melanomas. The superficial spreading subtype, younger patient age, thinner tumor, intermittent solar exposure, and early stage were significant predictors of a melanoma being histologically associated with a nevus. The appearance of a nevus associated with a melanoma predicted better recurrence-free survival compared with de novo melanomas. Although acral lentiginous melanomas (70.9%) constituted the most common histologic subtype, only 9.6% of the acral lentiginous melanomas were associated with a nevus. Furthermore, there was no statistically significant difference between the nevus-associated and de novo acral lentiginous melanomas with regard to clinicopathological factors and survival. In conclusion, nevus-associated melanomas were uncommon among acral lentiginous melanomas. Relatedly, because over half of all melanomas in Asians are acral lentiginous melanomas, Asians are less likely than Caucasians to have nevus-associated melanomas.</p></div

Clinical and histologic characteristics in acral lentigious melanomas.

<p>Clinical and histologic characteristics in acral lentigious melanomas.</p

Kaplan-Meier curves of survival for 73 patients with primary acral lentiginous melanomas.

<p>No overall (A), distant metastasis-free survival (B), and recurrence-free survival (C) differences were found between patients with de novo and nevus-associated acral lentiginous melanomas (<i>p</i> = 0.168, 0.159, and 0.091, respectively, log rank test).</p

Univariate and multivariate analysis of risk factors associated with overall survival.

<p>Univariate and multivariate analysis of risk factors associated with overall survival.</p