DNA extraction from paraffin embedded colorectal carcinoma samples: A comparison study of manual vs automated methods,using four commercially kits

BACKGROUND Nucleic acid isolation from formalin-fixed, paraffin-embedded tissue(FFPET)samples is a daily routine in molecular pathology laboratories, but extraction from FFPET is not always easily achieved. Choosing the right extraction technique is key for further examinations.AIM To compare the performance of four commercially available kits used for DNA extraction in routine practice.METHODS DNA isolation was performed on 46 randomly selected formalin-fixed, paraffinembedded(FFPE) colorectal adenocarcinoma(CRC) surgical specimens. Four commercially available extraction kits were used: two for manual DNA extraction(the Pure Link Genomic DNA Mini Kit from Invitrogen and the High Pure FFPE DNA Isolation Kit from Roche) and two for automated DNA extraction(the i Prep Genomic DNA Kit from Invitrogen and the Magna Pure LC DNA Isolation Kit from Roche). The DNA concentration and quality(odds ratio) among the four systems were compared. The results were correlated with the clinicopathological aspects of CRC cases: age, gender, localization, macro-and microscopic features,lymph node metastases, and the lymph node ratio.RESULTS The highest DNA concentration was obtained using the manual kits: 157.24 ±62.99 ng/μL for the Pure Link Genomic DNA Mini Kit and 86.64 ng/μL± 43.84 for the High Pure FFPE DNA Isolation Kit(P < 0.0001). Lower concentrations were obtained with automated systems: 20.39 ± 21.19 ng/μL for the Magna Pure LC DNA Isolation Kit and 8.722 ± 6.408 ng/μL for the i Prep Genomic DNA Kit,with differences between the systems used(P < 0.0001). The comparison between age, gender, tumor localization, pT or pN stage and the lymph node ratio indicated no statistically significant difference in DNA concentration using any of the nucleic acid isolation kits. DNA concentration was influenced by the macroscopic features and grade of differentiation. A higher DNA concentration was obtained for well-differentiated polypoid colorectal adenocarcinomas(CRCs), compared with undifferentiated ulcero-infiltrative carcinomas,irrespective of the kit used.CONCLUSION For research or diagnosis that needs high DNA concentrations, manual methods of DNA isolation should be used. A higher amount of DNA can be obtained from polypoid-type differentiated CRCs. Automated systems confer comfort and a lower amount of DNA that is, however, sufficient for classic polymerase chain reaction(PCR) and real-time quantitative PCR molecular examinations. All four commercially available kits can be successfully used in daily practice.

Exploration of Methods for Preparation of Cattle Eyeball Paraffin Sections

[Objective] This study aimed to establish a new method for preparing paraffin sections of cattle eyebal s. [Method] The conventional method was used to prepare paraffin sections for cattle eyebal s in the control and a new method termed＆quot;opening a window on cornea and refixation＆quot; was used to prepare paraffin sections for cattle eyebal s in the treatment group. [Result] After the prepared specimens in the treatment group were fixed, it could be macroscopical y observed that retina and choroid were closely connected, with detachment occurring at a smal portion be-tween the two. According to the paraffin sections, it was microscopical y observed that the continuity of trabecular meshwork was intact, as wel as the continuity be-tween different layers of eyebal wal , without detachment between them, no retinal detachment, no shrinkage of each layer of tissue cells. [Conclusion] This study pro-vides a foundation for the basic research and pathological study of eyebal s.

The Embedding Method for Nonsmooth Equations

In this paper. we present a class of' embedding methods for nonsmooth equations. Under suitable conditions, we Prove that there exists a homotopy solution curve, which is Unique and continuous. We also prove that the solution curve is singlcvalue-d with respect to the homotopy parameter. Then we construct all efficient algorithm for this class of equations and prove its convcrgcnce. Filially, we apply the algorithm to the nonlinear complementarity problem. The numerical results show that tile algorithm is satisfacotry.

A Numerical Embedding Method for Solving the Nonlinear Optimization Problem

A numerical embedding method was proposed for solving the nonlinear optimization problem. By using the nonsmooth theory, the existence and the continuation of the following path for the corresponding homotopy equations were proved. Therefore the basic theory for the algorithm of the numerical embedding method for solving the non-linear optimization problem was established. Based on the theoretical results, a numerical embedding algorithm was designed for solving the nonlinear optimization problem, and prove its convergence carefully. Numerical experiments show that the algorithm is effective.

A Decision Support Framework for the Choice of Languages and Methods for the Design of Real Time Embedded Systems

The choice of methods or design languages is a crucial phase in the development of systems and software, also for real time and embedded systems. An open question that remains in the design of these types of systems is to build a method, or to choose one among those existing, capable to cover the life cycle of a project, and particularly the development phases. This article contributes to answer the question, by proposing an approach based on a multi-criteria comparative study, of few languages and methods dedicated to the design of real time and embedded systems. The underlying objective of this work is to present to designers a wide range of approaches, and elements that can guide their choices. In order to reach this goal, we propose different comparison criteria. Each criterion is divided into sub-criteria, so that the designers can refine their choices according to the qualities they prefer and wish to have in the method or language. We also define a rating scale which is used to assess the retained languages and methods. The scores obtained from this assessment are presented in tables, one table per criterion, followed by a summary table giving the overall scores. Graphics built from these tables are provided and intend to facilitate the judgement and thus the choice of the designers.

A Paraffin Sectioning Method Without Using the Microtome and an Analysis Method for Optical Information of the Plant Anatomical Digital Photographs

[Objective] The research aimed to study a paraffin method without using the microtome,and also introduced an analysis method for optical information of the plant anatomical digital photographs.[Method] The plant material softened or not was embedded in paraffin according to the paraffin method.Cut the thin paraffin sections from the paraffin block with a sharp two-sided blade under anatomical lens.The thin material sections rolled up when they were cut off.Took the section rolls to a slide,and then heated them to melt the paraffin section roll.When the paraffin melted,the sections of plant material were rolled out.After the common or simplified procedures of staining and mounting,the preparations were finished.When an anatomical digital photograph was processed,copy it into the word file and two copies of the original photograph were obtained.One copy was selected to make it to be a negative photograph,and then press the key ＂Press Screen＂ to copy the screen frame.After it was copied into the word file,cut of the unnecessary parts and other operations were carried out,then processed photograph was obtained.[Results] The anatomical preparation for research was gotten.The analyzed digital photograph of the leaf structure of Salix matsudana var.matsudana f.tortuosa has some a three-dimensional effect,and the different leaf structures and cells,e.g.cuticle,cell wall,protoplast,vein,etc.can be identified easily.[Conclusion]The paraffin method without using the microtome has advantages of low cost and higher efficiency,which could be applied by the beginner or in the time without a microtome to be used.The analysis of the plant anatomical digital photographs can acquire more structural information than the original digital photographs,which shows the potentiality and prospects of the optical information analysis of the microscopic imagery and its digital photograph.

Real-time embedded software testing method based on extended finite state machine

The reliability of real-time embedded software directly determines the reliability of the whole real-time embedded sys- tem, and the effective software testing is an important way to ensure software quality and reliability. Based on the analysis of the characteristics of real-time embedded software, the formal method is introduced into the real-time embedded software testing field and the real-time extended finite state machine （RT-EFSM） model is studied firstly. Then, the time zone division method of real-time embedded system is presented and the definition and description methods of time-constrained transition equivalence class （timeCTEC） are presented. Furthermore, the approaches of the testing sequence and test case generation are put forward. Finally, the proposed method is applied to a typical avionics real- time embedded software testing practice and the examples of the timeCTEC, testing sequences and test cases are given. With the analysis of the testing result, the application verification shows that the proposed method can effectively describe the real-time embedded software state transition characteristics and real-time requirements and play the advantages of the formal methods in accuracy, effectiveness and the automation supporting. Combined with the testing platform, the real-time, closed loop and automated simulation testing for real-time embedded software can be realized effectively.

FORMATION ENTHALPIES FOR FCC METAL BASED BINARY ALLOYS BY EMBEDDED ATOM METHOD

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High-pressure phonon dispersion of copper by using the modified analytic embedded atom method

By using the Born-von Kfirmfin theory of lattice dynamics and the modified analytic embedded atom method, we reproduce the experimental results of the phonon dispersion in fcc metal Cu at zero pressure along three high symmetry directions and four oft-symmetry directions, and then simulate the phonon dispersion curves of Cu at high pressures of 50, 100, and 150 GPa. The results show that the shapes of dispersion curves at high pressures are very similar to that at zero pressure. All the vibration frequencies of Cu in all vibration branches at high pressures are larger than the results at zero pressure, and increase correspondingly as pressure reaches 50, 100, and 150 GPa sequentially. Moreover, on the basis of phonon dispersion, we calculate the values of specific heat of Cu at different pressures. The prediction of thermodynamic quantities lays a significant foundation for guiding and judging experiments of thermodynamic properties of solids under high pressures.

Detection of BCL2-IGHrearrangement on paraffin-embedded tissue sections obtained from a small submucosal tumor of the rectum in a patient with recurrent follicular lymphoma

A 59-year-old woman was admitted to our hospital because of recurrent follicular lymphoma(FL).Colonoscopic examination revealed a rectal submucosal tumor(SMT)without any erosions and ulcers.In this patient,it was difficult to distinguish non-Hodgkin's lymphoma(NHL)invasion from other disorders of the colon including carcinoid tumor merely based on endoscopic findings.Histopathologic and immunohistochemical studies on biopsy specimens showed an infiltration of atypical lymphocytes that were positive for CD20 and BCL2 but negative for UCHL-1.Fluorescence in situ hybridization on paraffin-embedded tissue sections (T-FISH)identified a translocation of BCL2 with IGHgene. Based on these findings,the tumor was defined as an invasion of FL.T-FISH method is useful for the detection of a monoclonality of atypical lymphocytes in an SMT of the gastrointestinal tract,and particularly for the detection of chromosomal translocations specific to lymphoma subtypes.

AN EMBEDDED BOUNDARY METHOD FOR ELLIPTIC AND PARABOLIC PROBLEMS WITH INTERFACES AND APPLICATION TO MULTI-MATERIAL SYSTEMS WITH PHASE TRANSITIONS

The embedded boundary method for solving elliptic and parabolic problems in geometrically complex domains using Cartesian meshes by Johansen and Colella （1998, J. Comput. Phys. 147, 60） has been extended for elliptic and parabolic problems with interior boundaries or interfaces of discontinuities of material properties or solutions. Second order accuracy is achieved in space and time for both stationary and moving interface problems. The method is conservative for elliptic and parabolic problems with fixed interfaces. Based on this method, a front tracking algorithm for the Stefan problem has been developed. The accuracy of the method is measured through comparison with exact solution to a two-dimensional Stefan problem. The algorithm has been used for the study of melting and solidification problems.

A New Method of Embedded Fourth Order with Four Stages to Study Raster CNN Simulation

A new Runge-Kutta （PK） fourth order with four stages embedded method with error control is presentea m this paper for raster simulation in cellular neural network （CNN） environment. Through versatile algorithm, single layer/raster CNN array is implemented by incorporating the proposed technique. Simulation results have been obtained, and comparison has also been carried out to show the efficiency of the proposed numerical integration algorithm. The analytic expressions for local truncation error and global truncation error are derived. It is seen that the RK-embedded root mean square outperforms the RK-embedded Heronian mean and RK-embedded harmonic mean.

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.

Chen’s lattice inversion embedded-atom method for Ni-Al alloy

The structural properties, the enthalpies of formation, and the mechanical properties of some Ni-Al intermetallic compounds （NiAl, Ni3Al, NiAl3, Ni5Al3, Ni3Al4） are studied by using Chen＇s lattice inversion embedded-atom method （CLI-EAM）. Our calculated lattice parameters and cohesive energies of Ni-A1 compounds are consistent with the experimental and the other EAM results. The results of enthalpy of formation indicate a strong chemical interaction between Ni and Al in the intermetallic compounds. Through analyzing the alloy elastic constants, we find that all the Ni-Al intermetallic compounds discussed are mechanically stable. The bulk moduli of the compounds increase with the increasing Ni concentration. Our results also suggest that NiAl, Ni3Al, NiAl3, and Ni5Al3 are ductile materials with lower ratios of shear modulus to bulk modulus; while Ni3Al4 is brittle with a higher ratio.

Structural defects in L1_0 FePt by modified analytic embedded-atom method

The structural defects of L10 FePt are investigated by the molecular dynamics (MD) with a modified analytic embedded-atom method (MAEAM). The L10 ordered structure of FePt is relaxed from a trial fcc structure. The defect formation energies are calculated. The vacancy formation energies of Fe and Pt are 1.89 eV and 2.11 eV respectively. The antisite formation energy of Fe in Pt sublattice is 0.35 eV. The antisite formation energy of Pt in Fe sublattice is 0.09 eV. The tendency of the vacancy formation energy is in agreement with other calculation. The point defect structure types are Pt antisite in rich-Pt side and Fe antisite in rich-Fe side.

Seismic analysis of cantilever earth retaining walls embedded in dry sand by simplified approaches and finite element method

In engineering practice simplified methods are essential to the seismic design of embedded earth retaining walls,as fullydynamic numerical analyses are costly,time-consuming and require specific expertise.Recently developed pseudostatic methods provide earth stresses and internal forces,even in those cases in which the strength of the soil surrounding the structure is not entirely mobilised.Semiempirical correlations or Newmark sliding block method provide an estimate of earthquake-induced permanent displacements.However,the use of these methods is hindered by uncertainties in the evaluation of a few input parameters,affecting the reliability of the methods.This study uses 1 D site response analyses and 2 D fully-dynamic finite element analyses to show that simplified methods can provide a reasonable estimate of the maximum bending moment and permanent displacements for stiff cantilever walls embedded in uniform sand,providing that a few input parameters are evaluated through semiempirical correlations and a simple 1 D site response analysis.

The ternary Ni–Al–Co embedded-atom-method potential for γ/γ Ni-based single-crystal superalloys: Construction and application

An Ni-AI-Co system embedded-atom-method potential is constructed for the γ（Ni）/γ＇（Ni3A1） superalloy based on experiments and first-principles calculations. The stacking fault energies （SFEs） of the Ni（Co, A1） random solid solutions are calculated as a function of the concentrations of Co and A1. The calculated SFEs decrease with increasing concentrations of Co and A1, which is consistent with the experimental results. The embedding energy term in the present potential has an important influence on the SFEs of the random solid solutions. The cross-slip processes of a screw dislocation in homogenous Ni（Co） solid solutions are simulated using the present potential and the nudged elastic band method. The cross-slip activation energies increase with increasing Co concentration, which implies that the creep resistance of γ（Ni） may be improved by the addition of Co.

Trefoil Factor 3 (TFF3) mRNA Expression Level in Follicular Thyroid Tumors Using Formalin-Fixed, Paraffin-Embedded (FFPE) Blocks

Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preoperative fine needle aspiration cytology, and may not be judged unanimously on permanent sections even among experienced pathologists. Determination of molecular-genetic factors such as trefoil factor 3 (TFF3) mRNA in the follicular thyroid tumors may be useful aid to improve the accuracy of diagnosis, though it is considered to be unstable and relatively low concentrated genetic substance. Purpose of our study is to investigate expression level of TFF3 mRNA of thyroid follicular tumors using formalin-fixed, paraffin-embedded (FFPE) tissue. Methods: Study population included FFPE sections from 19 FTC cases, 20 FTA cases, 11 adenomatous goiter (G) cases and 12 samples of normal thyroid tissue (N) adjacent to thyroid tumors. RNeasy FFPE kit was used for extraction of total RNA. Purification and concentration values were determined by spectrophotometer. Extracted RNA was used for cDNA synthesis in reverse transcription. Synthesized cDNA subsequently proceeded for relative quantification of TFF3 mRNA by RT-qPCR using TFF3 primers. Glyceroldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthin phosphorobosyltransferase1 (HPRT1) were used as control genes. The mean and standard deviation of TFF3 mRNA expression level were analyzed by software Multiplate RQ. Results: Extraction by the FFPE kit yielded high concentration of RNA in all cases. Purification values were 1.8 in average. Concentration values were significantly higher in FTC and FTA relative to G and N tissues, possibly due to high density of thyrocytes in the samples. Relative quantification of TFF3 mRNA expression level showed broad ranges both in FTC and FTA, while the analyses in G and N tissues indicated narrow ranges. Conclusion: FFPE tissues from thyroid follicular tumors can be used for measurement of unstable and low concentrated genetic substances such as TFF3 mRNA. Its diagnostic value yet remains to be determined.

Construction of embedded-atom-method interatomic potentials for alkaline metals (Li,Na,and K) by lattice inversion

The lattice-inversion embedded-atom-method interatomic potential developed previously by us is extended to alkaline metals including Li,Na,and K.It is found that considering interatomic interactions between neighboring atoms of an appropriate distance is a matter of great significance in constructing accurate embedded-atom-method interatomic potentials,especially for the prediction of surface energy.The lattice-inversion embedded-atom-method interatomic potentials for Li,Na,and K are successfully constructed by taking the fourth-neighbor atoms into consideration.These angular-independent potentials markedly promote the accuracy of predicted surface energies,which agree well with experimental results.In addition,the predicted structural stability,elastic constants,formation and migration energies of vacancy,and activation energy of vacancy diffusion are in good agreement with available experimental data and first-principles calculations,and the equilibrium condition is satisfied.

Simulation of He Behavior in Metals With Embedded Atom Method

The embedded-atom method(EAM)is used to study the behavior of helium in meta-ls.By fitting the measured parameters such as the activation energy and the heat of solution,the EAM potentials of helium in nickel are extracted.Based upon the EAM potentials,thebinding energy and the self-trapping of helium in nickel are investigated with molecular dynam-ics simulation.