Molecular and Immunological Techniques

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Questions and Answers

Which type of antibody is specifically produced from a single clone of cells?

Polyclonal antibodies

Recombinant antibodies

Avidin antibodies

Monoclonal antibodies (correct)

What is the primary function of immunocytochemistry?

To measure cell viability

To quantify the number of cells in a sample

To visualize protein localization in cells (correct)

To amplify DNA segments

In flow cytometry, what does light scatter correlate with?

Cell division rate

Protein synthesis rate

Cell size and granularity (correct)

Gene expression levels

Which molecular technique is primarily used for the amplification of specific DNA sequences?

Polymerase Chain Reaction (PCR) Signup and view all the answers

Which assay is commonly used for detecting specific proteins in a sample through their binding to antibodies?

Enzyme-linked immunosorbent assay (ELISA) Signup and view all the answers

What type of analysis can mass spectrometry provide in the context of proteomics?

Identification of protein composition Signup and view all the answers

What is the purpose of using specific primary antibodies in immunostaining techniques?

To bind and identify specific antigens Signup and view all the answers

What directly influences the level of deflection in mass spectrometry?

The mass and charge of ions Signup and view all the answers

What information can be derived from the deflection patterns observed in mass spectrometry?

The mass/charge ratio of the ions Signup and view all the answers

In mass spectrometry, why is the mass/charge ratio important?

It indicates the identity of the ions Signup and view all the answers

Which of the following aspects is least likely to affect the deflection of ions in mass spectrometry?

The size of the ion source Signup and view all the answers

During the mass spectrometry process, the detection of ions involves which of the following?

Capturing ions based on their mass and charge Signup and view all the answers

What can flow cytometry measure in relation to cell populations?

Cell size and granularity Signup and view all the answers

Which of the following is NOT a limitation of flow cytometry?

Limited particle measurement range Signup and view all the answers

What is the main purpose of Fluorescence-Activated Cell Sorting (FACs)?

To sort heterogeneous cell mixtures based on light scattering Signup and view all the answers

What is a key feature of ELISA in protein detection?

Antibody specific binding for quantification Signup and view all the answers

Which additional techniques enhance internal protein detection in ELISA?

Centrifugation, lysis, and fractionation Signup and view all the answers

In situ hybridization (ISH) is distinct from immunohistochemistry for which reason?

ISH localizes nucleic acid sequences Signup and view all the answers

What is a characteristic of modern multiplexing techniques used in ELISA?

Multiple targets in small sample volume Signup and view all the answers

Which particle size range can be measured by flow cytometry?

0.2 to 150 µm Signup and view all the answers

What is the purpose of bead assays in flow cytometry?

To measure cellular secretion Signup and view all the answers

What is the main purpose of in situ hybridisation?

To localize gene expression within a cellular environment Signup and view all the answers

What is a significant advancement of RT-qPCR compared to traditional PCR?

The capacity for real-time tracking of amplification products Signup and view all the answers

What is a primary limitation when using PCR?

Non-specific primer binding leading to multiple product amplification Signup and view all the answers

Which type of gel is typically used for DNA electrophoresis?

Agarose gel Signup and view all the answers

What method is used to isolate mRNA before reverse transcription in RT-qPCR?

RNA extraction techniques Signup and view all the answers

Which of the following best explains the role of a transcription binding dye in RT-qPCR?

It tracks and quantitatively compares increases in amplification Signup and view all the answers

What is the primary purpose of using electrophoresis in molecular biology?

To separate molecules based on their size and charge Signup and view all the answers

What type of analysis can PCR products be subjected to after amplification?

Electrophoresis and probing Signup and view all the answers

What can lead to issues with PCR amplification regarding the reference gene?

Selection of a reliable reference gene depends on multiple factors Signup and view all the answers

What is a typical dye used to visualize DNA during electrophoresis?

Ethidium bromide Signup and view all the answers

What is the main purpose of blotting techniques in electrophoresis?

To transfer separated nucleic acids or proteins onto a membrane Signup and view all the answers

Which method is primarily used for the quantitative analysis of proteins?

Western blotting Signup and view all the answers

What are common complications associated with Western blot analysis?

Poor sample preparation and non-specific antibody binding Signup and view all the answers

How does mass spectrometry characterize molecules?

By the mass to charge ratio Signup and view all the answers

What is the significance of unique signatures in mass spectrometry?

They enable comparisons to a known library. Signup and view all the answers

Which mass spectrometry technique is characterized by sample vaporization and ionization?

MALDI-TOF Signup and view all the answers

In the context of proteomics, what can mass spectrometry provide insights into?

Protein folding, structure, and function Signup and view all the answers

What type of molecules can be specifically targeted through labeling in mass spectrometry?

Phosphoproteome Signup and view all the answers

What is a common limitation of detection systems used in blotting techniques?

Inability to detect proteins at low concentrations Signup and view all the answers

What type of analysis does proteomic profiling perform with mass spectrometry?

Profiling of whole cell, intracellular or extracellular proteins Signup and view all the answers

Study Notes

Molecular and Immunological Investigation Techniques: A Survival Guide

This guide is for Year 2 students at the University of Plymouth, Peninsula Dental School.
The contact person for this guide is Charlotte Illsley.
Contact email: [email protected]
Contact details: PSQ C505
Learning outcomes for the session include: Recognizing relevant laboratory techniques for informed diagnosis and treatment planning, debating results with other biomedical professionals, staying current with scientific developments and evidence-based research techniques.

Why Learn Molecular and Immunological Investigative Techniques?

These techniques are important for understanding biological processes and their connections to diagnosis and treatment in a wide range of disciplines, including dentistry.

Targets – Where, What and Why

The different levels of biological organization (tissue, cellular, intracellular, genomic), including their potential causes and/or effects, are relevant for analysis using specific techniques
Techniques help determine if transcription, translation, secretion, expression or aggregation is involved.
The appropriate tool(s) for the investigation are crucial to achieve reliable results.

Molecular and Immunological Investigation Techniques

Nucleic acids
- In situ hybridization
- Polymerase Chain Reaction (PCR)
- Electrophoresis (e.g., Southern and Northern blots)
- Mass Spectrometry (RNA and DNA sequencing)
Proteins
- Immunostaining
- Flow cytometry
- Fluorescence-Activated Cell Sorting (FACs)
- Enzyme-linked immunosorbent assay (ELISA)
- Electrophoresis (Western blot)
- Mass Spectrometry (Proteomics)

Immunostaining

Immunocytochemistry
Immunohistochemistry
Immunofluorescence

Immunocytochemistry

Immunostaining technique used to visualize the localization of specific proteins/antigens
Uses primary antibodies that specifically bind to the target proteins
Secondary antibodies with associated fluorophores are conjugated to identify the target protein in cells

Antibodies

Polyclonal antibodies: derived from a single immune response to a given antigen. Multiple different antibodies are made against a single antigen
Monoclonal antibodies: produced from a single clone of immune-cells. All generated antibodies are identical.
Recombinant antibodies: produced synthetically in a lab, such as by genetically engineering antibodies or their fragments to function as a new antibody.

Flow Cytometry

Measures characteristics of cells (size, granularity, surface markers e.g. CD4, CD8)
Surface markers can classify cell types (e.g., T-cells)
Internalization, proliferation, survival, cytokine expression, chemokines and kinases can also be measured
Limitations include non-specific binding, signal balance/masking, fluorescent signal/wavelength crossover.

Fluorescence-Activated Cell Sorting (FACS)

Sorts cells based on their light-scattering and fluorescent characteristics
It separates heterogeneous mixtures of cells for further study.

ELISA (Enzyme-linked immunosorbent assay)

Measures protein expression or secretion by use of antibody-specific binding
Additional preparation steps like centrifuging, fractionation and lysis are sometimes needed to isolate targets in suspension
Multiplexing techniques allow for the detection of multiple targets using smaller sample volume
Quantification is determined using a comparison between the unknown sample and a known standard curve

In situ Hybridization (ISH)

A technique to locate specific nucleic acids (DNA or RNA) on chromosomes or in tissues
A probe that complements the target nucleic acid hybridizes to the target sequence.
The probe can then be visualized.
It complements immunohistochemistry by focusing on nucleic acids

FISH (Fluorescent In Situ Hybridization)

Variation of ISH using fluorescent probes to locate DNA or RNA within cells.
Denaturation and hybridization of the probe.
Visualization of hybridization using an epifluorescent microscope.

PCR (Polymerase Chain Reaction)

Method for amplifying DNA or RNA sequences using complementary primers
Basic PCR, RT-qPCR, and quantitative PCR allow real-time tracking of amplification products, allowing for comparative analysis of gene expression

Reverse Transcription – Quantitative Polymerase Chain Reaction (RT-qPCR)

Method to measure mRNA levels by reverse transcribing mRNA to cDNA, followed by PCR.
A binding dye tracks amplification, providing quantitative comparisons relative to a reference gene.

PCR Limitations

Susceptibility to contamination.
Non-specific primer binding.
Self-complementary primer binding/folding.
Selection of a reliable reference gene relies on multiple factors.

Electrophoresis

Separates molecules (DNA, RNA, proteins) based on size and charge.
Agarose and polyacrylamide gels are commonly used.
Post-gel analysis includes methods like staining (DNA - ethidium bromide; proteins- Coomassie blue, silver nitrate, Spyro ruby) and blotting (Western blot, Southern blot, or Northern blot).

Mass Spectrometry (MS)

Characterizes molecules by mass-to-charge ratio.
Provides unique molecular signatures for comparisons.
Can be used to profile proteins (proteomics) on whole cells, intracellular or extracellular levels.
Labeling can allow for specific target proteins i.e., phosphoproteome.

Microscopy

Light microscopy (various types), confocal microscopy, and electron microscopy can be used to visualize cells, tissues and organelles at various levels.
Fluorescent in situ hybridization is a useful method to locate mRNA in cells.
Electron microscopy involves using electrons instead of light, allowing for observation of finer details.

Summary

The presented techniques provide a comprehensive approach to studying molecular and immunological processes.
Various techniques are frequently combined to provide a comprehensive perspective.

Opportunities for Dental Research

Numerous opportunities exist for dental research in areas like oral health services research, oral cancer, periodontal research, 3D tissue engineering, dental nanomaterials, endotoxin/immunity, and oral microbiome research.

INSPIRE

For students interested in research careers, this program provides training and experience.

Intercalation

The university provides various opportunities for medical, dental, and veterinary students.

Studying That Suits You

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Description

This quiz examines critical molecular and immunological investigative techniques essential for Year 2 students at the Peninsula Dental School. Students will explore various laboratory techniques for diagnosis and treatment planning while debating scientific results with peers. Stay informed with current advancements and evidence-based practices relevant to dentistry.

Molecular and Immunological Techniques - Year 2 Dental

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