Mock grant application roleplay as an alternative to lab-based activities in molecular biology.

Many universities resort to online teaching due to COVID-19 pandemic. It is a challenging endeavor, especially in Molecular Biology courses that require lab access. Mock grant application roleplay is one alternative to lab-based activities. Students are engaged in three aspects: (i) targeted literature review, (ii) research proposal writing and (iii) 5-min project pitching. The design of this module is flexible and, other lab-based courses can adopt it. This module encourages undergraduate students to explore the lab techniques they learnt and concisely present their research proposal.

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 °C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

[Immunohistochemistry using clone VE1 is an economic, specific and sensitive method for detecting the presence of BRAFV600E mutations in melanoma]. / L'immunohistochimie (clone VE1) est une méthode économique, spécifique et sensible pour détecter la présence d'une mutation BRAFV600E dans le mélanome.

BACKGROUND: Determination of BRAF mutation status is mandatory in the management of patients with inoperable stage IIIC or stage IV melanoma. Currently, molecular biology (MB) has been validated for detecting the presence of BRAF mutations. OBJECTIVE: To compare the sensitivity, specificity and cost of immunohistochemistry (IHC) (clone VE1) versus BM methods (qPCR and Sanger sequencing). PATIENTS AND METHODS: All the samples for which BRAF mutation status was requested between March 2013 and February 2015 at the cellular and molecular analysis laboratory of the Angers Hospital were included retrospectively and consecutively. The IHC (clone VE1) and BM analyses were performed with the same formalin-fixed paraffin embedded tumour samples. The cost of these two methods was determined on the basis of the cost for the French Health Insurance. RESULTS: Two hundred and seven samples were subjected to a determination of BRAF mutational status in IHC and BM. Only one sample was discordant between these two methods (positive in IHC, negative in BM). The sensitivity and specificity of the IHC was 100% and 99.25% respectively. The ratio of the cost of IHC/BM testing was 1:2.1. CONCLUSION: IHC (clone VE1) is a specific, sensitive and economic method for determining BRAFV600E mutation status. Nevertheless, this method must be validated in order to be integrated into a decisional algorithm, alongside BM methods, to determine whether targeted BRAF-inhibitor therapy is indicated.

Cost-effectiveness of methods in personalized medicine. Results of a decision-analytic model in patients with acute myeloid leukemia with normal karyotype.

BACKGROUND: During the last years, molecular genetic data are increasingly used as prognostic and predictive factors in acute myeloid leukemia (AML). The molecular genetic profile permits a rapid risk categorization and beyond that a prediction of differential treatment efficacy of post-remission chemotherapy versus an allogeneic hematopoietic cell transplantation (HCT) in specific subgroups. METHODS: The aim of this study was to evaluate cost-effectiveness of two different strategies of risk categorization (conventional cytogenetic diagnostics (CCD) versus molecular genetic diagnostics (MGD)) in patients with AML, using a decision-analytic state-transition model. The model is run as (Monte Carlo) microsimulation in which individuals pass through in cycles with a cycle length of one month and a time horizon of ten years. FINDINGS: Results show that on average, individuals within the MGD group generated about US$ 32,000 higher costs but survived about seven months longer than individuals within the CCD group. This leads to an Incremental Cost-Effectiveness Ratio (ICER) of about US$ 4928 per survived month. INTERPRETATION: With a GDP (Gross Domestic Product) of US$ 26,467 (€ 33,630) per capita in Germany in 2012, the base-case ICER of US$ 4928 per survived month projected to US$ 59,136 per survived year is in between the simple GDP and the three times GDP per capita.

An easy, quick and cheap high-throughput method for yeast DNA extraction from microwell plates.

We herein present a high-throughput and cheap method for yeast DNA isolation in a 96 well microplate. About 1500 yeast isolates can be processed within one working day and final DNA concentrations are suitable for direct application in PCR-based molecular typing methods.

Evaluation of a reliable and cost-effective method of DNA isolation for mouse genotyping.

Genotyping is commonly used to define specific gene alterations or the presence of transgenes in mice. This procedure is typically done using DNA isolated from mouse tail tissue. Although there are commercially available kits for tail DNA isolation, they can be time consuming and costly for routine genotyping. In this study, we describe a rapid, "crude" DNA isolation method using mouse tail tissue and compare it to a frequently used, commercially available kit in the genotyping of over 1,000 total mice from 8 genetic lines. Our genotyping results were obtained faster and less expensively but with the same success rate (Crude method: 97.7 %, Kit method: 98.4 %). To our knowledge, this is the first systematic study to compare the reliability of this crude DNA isolation method for mouse genotyping compared to a commercially available kit.

High-resolution melting analysis: a genotyping tool for population studies on Daphnia.

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour- and time-consuming. Here, we demonstrate high-resolution melting analysis (HRMA) as a cost-effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double-stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.

Rapid and cost-effective baculovirus sample preparation method as a viable alternative to conventional preparation for quantitative real-time PCR.

The increasing use of the baculovirus expression vector system (BEVS) has generated significant interest into techniques for quantifying baculovirus stocks. One method involves the use of quantitative real-time polymerase chain reaction (PCR). This study investigated simplifying baculovirus sample preparation for quantitative Real Time PCR to provide an alternative to current kit-based preparation methods. To achieve this goal, combinations of freeze/thaw cycles and Triton X-100 treatment were investigated. A treatment with only Triton X-100 was found to be sufficient to provide a simple, rapid and cheap alternative to kit-based preparation methods. This study also examined other factors such as primer choice to further examine the process of baculovirus quantitation by qPCR.

Comparison of genomic DNA extraction techniques from whole blood samples: a time, cost and quality evaluation study.

Genomic DNA obtained from patient whole blood samples is a key element for genomic research. Advantages and disadvantages, in terms of time-efficiency, cost-effectiveness and laboratory requirements, of procedures available to isolate nucleic acids need to be considered before choosing any particular method. These characteristics have not been fully evaluated for some laboratory techniques, such as the salting out method for DNA extraction, which has been excluded from comparison in different studies published to date. We compared three different protocols (a traditional salting out method, a modified salting out method and a commercially available kit method) to determine the most cost-effective and time-efficient method to extract DNA. We extracted genomic DNA from whole blood samples obtained from breast cancer patient volunteers and compared the results of the product obtained in terms of quantity (concentration of DNA extracted and DNA obtained per ml of blood used) and quality (260/280 ratio and polymerase chain reaction product amplification) of the obtained yield. On average, all three methods showed no statistically significant differences between the final result, but when we accounted for time and cost derived for each method, they showed very significant differences. The modified salting out method resulted in a seven- and twofold reduction in cost compared to the commercial kit and traditional salting out method, respectively and reduced time from 3 days to 1 hour compared to the traditional salting out method. This highlights a modified salting out method as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples.

[Detection of Staphylococcus aureus resistant to methicillin (MRSA) by molecular biology (Cepheid GeneXpert IL, GeneOhm BD, Roche LightCycler, Hyplex Evigene I2A) versus screening by culture: Economic and practical strategy for the laboratory]. / Dépistage de Staphylococcus aureus résistant à la méthicilline (SARM) par biologie moléculaire (Cepheid GeneXpert IL, GeneOhm BD, LightCycler Roche, Hyplex Evigene I2A) versus dépistage par culture : stratégie économico-pratique pour le laboratoire.

OBJECTIVES: Patients admitted in cardiac surgery and cardiac ICU at the Clinic Saint-Gatien (Tours) are screened for MRSA at the entrance by nasal swab and culture on blood agar and selective chromogenic medium made by addition of cefoxitin: BBL CHROMagar MRSA-II BD (result obtained at Day +1). We wanted to assess the molecular biology techniques available to obtain a result at day 0 for the majority of patients and to define an economic and practical strategy for the laboratory. TECHNIQUES: We studied four molecular biology techniques: Cepheid GeneXpert (Cepheid) GeneOhm (BD), LightCycler (Roche) and Hyplex (I2A). Upon reception, nasal swabs were treated by culture, considered as reference, and one of the techniques of molecular biology, according to the manufacturer's notice. We conducted four studies between April 2008 and February 2009 to obtain a significant sample for each of them. METHODS: By screening we mean a method that allows us to exclude MRSA carriage for patients waiting for surgery, and not to change patient management: for example, lack of isolation measures specific to entrance, no modification of antibiotic prophylaxis during surgery and no isolation measures in the immediate postoperative period. RESULTS: The criteria we considered for this evaluation were: (1) technician time: time to perform one or a series of sample(s) n=10 or more (about 2h for all techniques except GeneXpert 75min), level of skilled competences (no specific training for GeneXpert); (2) results: turnaround time (all molecular biology techniques), ease of reading and results interpretations (no specialized training required for GeneXpert), failure or not (12% of failure of internal controls for GeneOhm); (3) economic: cost for one or a series of sample(s) (n=10 or more), if we considered X as the reference culture cost (10 X Hyplex and LightCycler, 20 X and 40 X for GeneXpert GeneOhm); (4) NPV: 100% for GeneXpert and LightCycler. CONCLUSION: At same sensitivity, no technique, including culture, can solve alone our problem, which is: (1) get results at day 0 for batch of samples (n<10): all molecular biology techniques; (2) beyond 10 samples: LightCycler (Roche) automated or Hyplex (I2A) manual; (3) when the result at day 1 is sufficient, the use of chromogenic agar with a reading of less than 18h as BBL CHROMagar MRSA II (BD) remains the most economical; (4) to be sure that a patient admitted at Day 0, even at night's emergency, is not carrier of MRSA: only Cepheid GeneXpert technology (IL). Furthermore, Cepheid GeneXpert (IL) allows performing several tests in parallel. The rapidity of this system can help control the transmission and make better use of antibiotics.

An inexpensive and rapid method for extracting papilionoid genomic DNA from herbarium specimens.

Three DNA extraction protocols were compared for their ability to yield DNA from the leaves of herbarium specimens of nine species from nine genera of the Papilionoideae. We tested two protocols that use classic procedures for lysis and purification with cetyl trimethylammonium bromide (CTAB); a third protocol used a Nucleospin Plant kit. DNA obtained from all three procedures was quantified and tested by PCR. Test results indicated the superiority of one of the CTAB protocols. We made some modifications, developing a protocol that produced high-quality DNA from all nine species. The modification involved the use of a lower EDTA concentration (20 mM instead of 50 mM) and a higher beta-mercaptoethanol concentration (1% instead of 0.4%) in the extraction buffer. The modified protocol avoids the necessity for a second DNA precipitation step. This new CTAB protocol includes the use of 1.4 M NaCl, 20 mM EDTA and 1% beta-mercaptoethanol in the extraction; DNA precipitation time is reduced. A reduction in contaminating metabolites (such as PCR inhibitors) in the sample mixtures and lower costs for reagents are characteristics of this modified protocol; the cost of analysis per sample was lowered, compared to previous options. The quality of DNA was suitable for PCR amplification. This is a practical alternative to more difficult, time-consuming and expensive protocols.