Welcome to this exciting webinar: Optimize your PCR, where we will delve into the intricacies of this powerful molecular biology technique. Our speaker, Gabriel Almeida Alves, BSN, MS, PhD, will take us on a journey through the world of PCR, exploring its various components, steps, and troubleshooting methods.

Join us as we delve into the world of PCR and learn about:

• DNA Template and Purification: Learn about the importance of a pure and high-quality DNA sample for efficient PCR amplification.

• DNA Polymerases and Buffers: Explore the different types of DNA polymerases and their respective buffers that are commonly used in PCR reactions.

• Oligo Design - The Ideal Primers: Understand the critical role of primers in the PCR reaction and learn how to design the ideal primer set.

• Choosing the Right Plastics: Find out the significance of plastics in PCR and learn how to choose the appropriate plasticware for your experiment.

• Thermal Cyclers: Discover the different types of thermal cyclers and their respective advantages and limitations.

• Post-PCR Workflow - Electrophoresis: Learn about the crucial post-PCR analysis step of electrophoresis and how it helps us to visualize and analyze the amplified DNA product.

This webinar will provide you with an in-depth understanding of PCR, its various components, and their importance. You will also learn about common pitfalls of PCR and how to avoid or troubleshoot them. We hope that this webinar will be a valuable resource for both new and experienced researchers alike, and help you take your PCR experiments to the next level!

Learning Objectives:

1. DNA Template and Purification: Discuss the importance of a pure and high-quality DNA sample for efficient PCR amplification.

2. DNA Polymerases and Buffers: Explain the different types of DNA polymerases and their respective buffers that are commonly used in PCR reactions.

3. Thermal Cyclers: Discover the different types of thermal cyclers and their respective advantages and limitations.

Cell and gene therapies, such as chimeric antigen receptor T cell therapies (CAR-T), have led to some miraculous cures that are transforming the clinical landscape for treating certain types of tumors, such as B cell lymphomas and various blood cancers.  Despite its great promise, many barriers continue to limit the effectiveness of CAR-T therapies. One of the largest barriers is developing reliable potency assays for these cell-based medicines, which by nature are highly heterogeneous. An engineered lot will inevitably have a distribution of growth properties, cytolytic activity, gene expression patterns, and secretion profiles, and it is not clear which cell subpopulations are most correlated with positive clinical outcomes. Since each lot of drug uses a patient’s own cells, drug testing must be performed on every patient, which magnifies the need for reliable potency assays.  In this talk, I will summarize the current status and future directions of the field of quality control analysis of cell based medicines, while focusing on the emergence of single cell based functional screening tools that can measure the cytolytic properties of individual T cells.  In particular, I will summarize our recent results on a platform that we are developing at Celldom, which uses a high density microwell array and high throughput image analysis techniques to analyze the statistical distribution of T-cell cytolytic properties across a range of effector to target ratios (E:T).  I will also describe the applications of our functional single cell screening tools in other applications, such as BCR and TCR discovery, cell line development, and drug resistance testing for applications in patient stratification.  

Learning Objectives:

1. Discuss the different types of quality control metrics and manufacturing controls required for hetereogeneous cell based medicines.

2. Discuss the importance of multi-parameter methods for characterizing single effector cells in cocultures.

3. Describe the importance of quantifying rare cell responses in a sample, and their effect on clinical outcomes.

We have developed 3D-shaped hydrogel microparticle platforms to capture cells, as well as isolate and label their secretions. These “lab on a particle” systems enable sorting cells based on secreted products for the discovery of antibodies, the development of cell lines producing recombinant products, and the selection of functional cells for cell therapies. Each cell and its secreted products can be analyzed using widely available flow cytometers and single-cell sequencing instruments, promising to democratize single-cell technologies. I will discuss our latest results linking the transcriptomes of single cells to the secreted products they produce. I will cover two example applications of this secretion encoded single-cell sequencing (SEC-seq) workflow. In a first example we uncover gene networks associated with high secretion of immunoglobulin G in human plasma cells, one of the most highly secreting cell types in the body. In a second application we characterize a unique transcriptionally-defined cluster of mesenchymal stromal cells (MSCs) that secrete higher levels of vascular endothelial growth factor (VEGF). VEGF is a pro-regenerative growth factor that is thought to drive therapeutic benefit in MSC-based therapeutics. I will summarize the potential for nanovial technology to promote understanding and engineering of functional properties in single cells, and ultimately drive the next-generation of cell therapies.

Learning Objectives:

1. Discuss the importance of probing cellular functions, such as single-cell secretions.

2. Develop assays to probe single-cell function using nanovials.

3. Correlate the function of single cells to underlying gene transcripts using flow cytometry and single-cell sequencing.

The enzyme-linked immunosorbent assay (ELISA) is one of the most common techniques performed in both research and clinical laboratories to detect the molecules of interest in fluid samples. In this webinar, we will demonstrate the potential for automating common ELISA assays, including including a sandwich EIA for detection of human fibronectin (FN), a competitive ELISA detecting an antigen, and a competitive ELISA detecting an antibody, and a bead-based ELISA for quantifying SARS-CoV-2 antibodies. These ELISAs were automated on the Opentrons OT-2 in order to streamline the workflow of this labor intensive assay. We'll show suggested settings for incubations and signal development steps of each assay, as well as well as results comparing manual and automated techniques.

It is well known that laboratories often handle sensitive information. The negative consequences can be severe when there is a breach of data and confidentiality. Therefore, when choosing a good Laboratory Information Management System (LIMS), the security of the LIMS should be a primary consideration. 
Sensitive data is all confidential information stored, processed, or managed by a laboratory relating to a patient or client that should not be revealed to a third party and is only accessible to authorized users. It is necessary to safeguard the privacy and integrity of all sensitive data to avoid penalties, financial loss, or compromising an institution’s security.
 
Sensitive data can be categorized into four main categories: 

1. Protected Health Information (PHI)
2. Personally Identifiable Information (PII)
3. Commercial Information
4. Clinical Trials and Study Information
 
A LIMS can handle security in different ways. From assigning log-ins and passwords to each user to restricting access to information that does not apply to them. Also, a suitable LIMS should ensure that information remains secure in case of system failures or cyberattacks. 
 
 
In this talk, I will unveil five essential considerations that you should take into account when choosing a LIMS:

1. Secure Data Servers
2. Secure Login
3. Role-based Access
4. Captcha and Two-Factor Authentication
5. Data Encryption (in transmission & at rest)
 
Learning Objectives:

1. Discuss sensitive data handled by laboratories.
2. List measures you must take to prevent cyberattacks in your laboratory.
3. Explain how an informatics tool, such as a LIMS, can help secure sensitive data and prevent cyberattacks.
 

Digital Droplet PCR (ddPCR) is a method for performing digital PCR use microfluidic water-oil emulsion technology.  By utilizing microfluidics and fractionating DNA samples into nanoliter droplets , ddPCR enables absolute quantification of DNA.  In comparison to traditional qPCR, ddPCR has demonstrated improved precision and accuracy, better sensitivity, and increased signal-to-noise ratio.  ddPCR also allows for traditional qPCR techniques including different probe chemistries and multiplexing.  ddPCR has enabled and improved many applications including NIPT, pathogen detection, food safety analysis, and liquid biopsy.  

Learning Objectives:

1. Explain the fundamental mechanism of ddPCR and how Poisson correction enables absolute quantification of DNA.

2. Describe the advantages and disadvantages of ddPCR in relation to qPCR.

3. Recognize current applications of ddPCR in many different fields including diagnostics and food safety.

Regulated laboratories must comply with numerous quality requirements that are put in place by regulatory agencies to ensure that accurate data is being generated and reported. The vast majority of laboratories strive to generate high quality, accurate, affordable data. To achieve this, they acquire LIMS solutions and automation to help them in implementing quality measures, a few include, positive ID, SOPs (Standard Operating Procedures), Method Validation, known standards included in runs, equipment maintenance and calibration and provide team members specialized training. These requirements touch upon various processes throughout the sample lifecycle, and begin at Sample Login, where each sample must have a unique sample ID. To ensure that samples are properly labeled, without transcription errors, many laboratories leverage positive ID, such as 1-Dimensional or 2-Dimensional barcoding or RFID (Radio-Frequency Identification). Once samples are logged into the system, they must be managed, tests and QC assigned and tracked through sample disposal. To ensure quality, each step of the analysis is tracked, monitored and all chemicals, supplies and reagents are tracked and traced throughout the analysis process. Analytical instrumentation is monitored, managed and calibrated with all the results recorded. Instrument data is electronically imported to eliminate transcription errors and other controls and sensors are put in place to eliminate human errors. Restful APIs are utilized to accelerate communication with other enterprise systems. System auditing allows auditors to understand why results were altered and to see the number of audits over time. 

Learning Objectives:

1. Discuss the Role of a LIMS in a Quality Management System. 

2. Recognize that there are many other systems that are tightly integrated with LIMS, which scientists may not be aware or, which are available to further cost effectively facilitate data integrity. 

3. List the key features and functions of a LIMS. 

Preclinical research and development are the cornerstone of modern drug discovery. A variety of biological systems including in vitro, 3D cultures, and organoids are essential tools, with ultimately lead candidates being tested in animal models. Microfluidics has played a significant role in advancing research and providing high content data in in vitro biological systems. In this talk, I will discuss our laboratory’s effort to apply microfluidic approaches to perform high throughput phenotypic screening in C. elegans – a nematode that offers human-relevant biology and is suitable to microfluidic automation.  This in vivo model addresses the limitations of in vitro models, where tissue-level and multi-organ interactions are difficult to capture. Likewise, it alleviates the need for expensive and unsustainable testing in rodent models. Thus, C. elegans based phenotypic screening offers an attractive paradigm to re-think modern drug discovery. Moreover, with recent advances in transgenics, a variety of disease models have been engineered in C. elegans capturing relevant biology for neurodegeneration, muscle disorders and rare genetic diseases. I will also discuss, the use of microfluidics to conduct C. elegans based studies on the International Space Station with a focus on muscle health. The results from our studies show that whole-life in vivo data can be obtained using C. elegans and microfluidic automation. Our technology platform could be used to screen compound libraries for in vivo efficacy at a fraction of cost and time compared to rodent models.  

Learning Objectives:

1. Discuss the challenges of preclinical drug discovery.

2. List the advantages of C. elegans and microfluidics for in vivo screening.

3. Discuss the phenotypes that can be quantified in microfluidic in vivo screening.

We have developed an electronic method to screen cells for their phenotypic profile, which we call Node-Pore Sensing (NPS). NPS involves using a four-terminal measurement to measure the modulated current pulse caused by a cell transiting a microfluidic channel that has been segmented by a series of inserted nodes. By simply inserting between two nodes a straight “contraction” channel through which cells can squeeze, we can simultaneously measure a cell’s size, resistance to deformation, transverse deformation, and ability to recover from deformation. I will describe how we have used “mechano-NPS” to distinguish sublineages of primary human mammary epithelial cells and the chronological age groups (i.e. “young” vs. “old”) of women from which these cells were derived—all based on the mechanical properties of the cells measured. In addition to mechanophenotyping cells, I will show how we have performed label-free, immunophenotyping of cells using NPS. In this case, cell transit an NPS device in which different segments are functionalized with antibodies corresponding to different cell-surface epitopes. When a cell has a specific surface epitope that can interact specifically with the functionalized antibodies, the cell will transit slower in that segment vs. in a segment functionalized with a control. I will show how we have successfully immunophenotyped blasts found within the bone marrow of acute myeloid leukemia patients with NPS, and how we are now using a method called highthroughput DNA-directed patterning to functionalize our devices in a straightforward and efficient manner. Finally, I will also describe the development of the next-generation NPS platform which utilizes advanced signal processing algorithms—Barker and Gold codes—directly encoded in the NPS channels to thus achieve multiplexing and higher throughput.

Learning Objectives:

1. Explain how to utilize microfluidics to perform mechanophenotyping.

2. Discuss how to utilize mechanophenotyping to differentiate malignant vs. non-malignant cells and to assess the chronological age of women who’s cells are mechanophenotyped.

3. Discuss a new method of functionalizing antibodies on a substrate and how to perform label-free immunophenotyping.

Our lab has developed a novel platform for droplet sorting that can be used to sort enzymes by activity, and cells by metabolism. It is a passive technique that utilizes no excitation laser, detector, active sorter and related electronics, or fluorescent labels. It presents a robust way to sort enzyme libraries for screening and sort cancer cells by a defining characteristic, their aberrant metabolism. The technique also enables the isolation of activated T-cells prior to the display of cellular surface markers. The isolation of activated T-cells in minutes rather than hours can have broad usage as a biotechnology tool and for the study and selection for immunology and immunotherapy.

Learning Objectives:

1. Explain the droplet sorting mechanism of the SIFT technology.

2. Discuss the advantages and disadvantages of the SIFT technology.

3. Identify the type of samples that can be sorted with the SIFT technology.