RESEARCH TECHNIQUES MADE SIMPLE: ENZYME IMMUNOASSAY (EIA) and ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Q&A

The questions and answers below relate to the Research Techniques Made Simple article  “Enzyme Immunoassay (EIA) and Enzyme-Linked Immunosorbent Assay (ELISA),” published online with the September 2013 issue of JID.

 

Correct answers appear in bold underline.

 

Multiple Choice Questions & Answers:

 

1.    Which of the following molecule(s) can be detected by ELISA?

a)     proteins

b)     hormones

c)     antibodies

d)     all of the above

 

2.     What does a weak color signal in competitive ELISA represent?

a)     more antigen in the sample

b)     less antigen in the sample

c)     less antigen retained on the well

d)     both a and c

 

3.    Which of the following is immobilized on the microtiter well in sandwich ELISA?

a)    detection antibody

b)    sample

c)    capture antibody

d)    secondary antibody conjugated to an enzyme

 

4.    What is a major advantage of ELISA in comparison to other biological quantification techniques?

a)    detection of a molecule at a low concentration

b)    inexpensive

c)    low specificity

d)    easily available

RESEARCH TECHNIQUES MADE SIMPLE: NEXT GENERATION SEQUENCING Q&A

The questions and answers below relate to the Research Techniques Made Simple article  “Next Generation Sequencing: Methodology and Application,” published online with the August 2013 issue of JID.

Correct answers appear in bold underline

 

Multiple Choice Questions & Answers:

 

1.     The basic methodological steps of NGS include:

a)     Template preparation, emulsion PCR, sequencing, data analysis

b)     Template preparation, sequencing and imaging, data analysis

c)     Template amplification, sequencing and imaging, data analysis

d)     Template preparation, sequencing and imaging, alignment to a reference genome

e)     DNA fragmentation, sequencing, data analysis

 

 

2.     Advantages of targeted sequencing as opposed to full genome, exome, or transcriptome sequencing include:

a)     Affordable and efficient for quickly interrogating particular genomic regions of interest

b)     Provides a deeper coverage of genomic regions of interest

c)     Can be utilized in deciding a therapeutic plan of action for both germline and somatic cancers

d)     Detects and quantifies low-frequency variants such as rare drug-resistant viral mutations (e.g., HIV, HBV or microbial pathogens)

e)     All of the above

 

 

3.     Applications of NGS in medicine include:

a)     Detecting mutations that play a role in diseases such as cancer

b)     Identifying genes responsible for inherited skin diseases

c)     Determining RNA expression levels

d)     Identifying novel virulence factors through sequencing of bacterial and viral species

e)     All of the above

RESEARCH TECHNIQUES MADE SIMPLE: NORTH, SOUTH, OR EAST? BLOTTING TECHNIQUES

Below are questions and answers related to the Research Techniques Made Simple article from the July 2013 issue of JID, entitled “North, South,or East? Blotting Techniques” by M.W. Nicholas, M.D., Ph.D.1 and Kelly Nelson, M.D.2

1University of North Carolina at Chapel Hill Department of Dermatology and 2Duke University Department of Dermatology.

 

Questions  (correct answer is underlined)

 

1.       The target molecule type for Southern blotting is:

A      RNA

B      DNA

C      Protein

D      Lipids

 

2.       Limitations of the western blot technique include all of the following except:

A      Low specificity

B      Loss of antibody epitope with denaturing

C      Less accurate determination of quantity

D      Higher cost compared to ELISA

 

3.       Which of the following techniques is most commonly employed in modern research?

A      Southern blot

B      Northern blot

C      Western blot

D      Eastern blot

 

4.       Place the following blotting steps in order:  (Answer: C,A,D,B)

A      Transfer to membrane

B      Detect probe

C      Separate via gel electrophoresis

D      Treat with probe

RESEARCH TECHNIQUES MADE SIMPLE: GENOME-WIDE EPIGENETICS Q&A

The questions and answers below relate to the Research Techniques Made Simple (RTMS) entitled “ Genome-Wide Epigenetics” in the June 2013 issue of Journal of Investigative Dermatology.  To view the article, go to the RTMS web page.

 

Questions & Answers

 

1)          Heterochromatin refers to:

A.           Actively transcribed regions of DNA to which transcription factors actively bind

B.           The core structure around which 147 base pairs of DNA bind

C.           Constitutively closed and transcriptionally repressed areas of genome organization

D.           Areas of the genome marked by the histone modification H3K4me3

 

The correct answer is C:  Constitutively closed and transcriptionally repressed areas of genome organization.

 

 

 

2)          The major role of ChIP-seq experiments lies in:

A.           The ability to map DNA methylation patterns across the genome

B.           The ability to map the bound histone modifications and other proteins bound to chromatin across the genome

C.           The ability to analyze gene expression changes with changes in chromatin modifications

D.           The ability to associate single-nucleotide polymorphisms with various disease states

The correct answer is B:  The ability to map the bound histone modifications and other proteins bound to chromatin across the genome.

RESEARCH TECHNIQUES MADE SIMPLE: FLUORESCENCE IN SITU HYBRIDIZATION (FISH): Q&A

This quiz relates to the Research Techniques Made Simple article “FLUORESCENCE IN SITU HYBRIDIZATION (FISH)” published in the May 2013 issue of the Journal of Investigative Dermatology.

 

 

Questions:

 

 

1. What does FISH detect?

a. Protein structure abnormalities
b. Specific chromosome copy number aberrations
c. Presence of specific antigens
d. Presence of complement

 

 

2. Where does the FISH probe localize to?

a. Golgi apparatus
b. Cytoplasm
c. Cell membrane
d. Nucleus
3. What is the FISH probe composed of?

a. Proteins
b. Lipids
c. Carbohydrates
d. Nucleic acids

 

 

4. What is the maximum number of FISH probes that can be used in a single experiment?

a. Two
b. Three
c. Four
d. Five

 

 

 

Answers:


1.         The correct answer is b:  Specific chromosome copy number aberrations

2.         The correct answer is d:  Nucleus

3.         The correct answer is d:  Nucleic acids

4.         The correct answer is c:  Four

 

RESEARCH TECHNIQUES MADE SIMPLE: MICROARRAY Q&A

The questions and and answers below relate to the Research Techniques Made Simple entitled “Microarray Technique, Analysis and Applications in Dermatology” in the April 2013 issue of Journal of Investigative Dermatology.

 

 

1.  The term “array” in microarray refers to the arrangement of which of the following on the chip:

a. the probe
b. the target
c. the fluorophore
d. the antigen

The correct answer is a:  the probe

 

 

2. The most common types of probes used for microarrays are:

a. complementary DNA (cDNA)
b. single nucleotide polymorphisms (SNPs)
c.antibodies
d.double-stranded DNA

The correct answer is a: complementary DNA (cDNA)

 

 

3. Microarray analysis has been used to study

a. melanoma
b. psoriasis
c. cutaneous T-cell lymphoma
d. scleroderma
e. all of the above

The correct answer is e:  all of the above

 

 

 

RESEARCH TECHNIQUES MADE SIMPLE: Polymerase Chain Reaction (PCR) Q&A

The questions and answers below relate to the Research Techniques Made Simple article entitled “Polymerase Chain Reaction” published in the March 2013 issue of the Journal of Investigative Dermatology

 

Questions

 

1. Qualitative PCR and quantitative PCR provide information on ____________ and ________ respectively.

A. presence/absence of specific DNA product; how much of a specific DNA product is present

B. how much of a specific DNA product is present; presence/absence of DNA product

C. RNA; DNA

D. gene byproducts; RNA

 

Answer:  A. presence/absence of specific DNA product; how much of a specific DNA product is present. Qualitative PCR is used to detect the presence or absence of a specific DNA product.  Qualitative PCR is a good technique to use when PCR is performed for cloning purposes or for identification of a pathogen. On the other hand, quantitative PCR provides more information beyond just mere detection of DNA.  It is able to indicate how much of a specific DNA or gene is present in the sample.

 

 

2. The most widely used method of analysis of the PCR product is:

A. agarose gel electrophoresis

B. western blot

C. ELISA

D. FISH

 

Answer:  A. agarose gel electrophoresis. The most widely used method of analysis of the PCR product is via the use of the simple agarose gel electorophoresis.  The agarose gel electrophoresis is the easiest and most common method of visualizing and analyzing the PCR product.  It allows the determination of the presence and the size of the PCR product.   A predetermined set of DNA products with known sizes are ran on the gel as molecular markers to help determine the size of the product. The two other methods for visualizing the PCR products are staining of the amplified DNA product using a chemical dye such as ethidium bromide, which intercalates between the two strands of the duplex or labeling the PCR primers or nucleotides with fluorescent dyes (fluorophores) prior to PCR amplification.

 

 

3. A major advantage of using PCR as compared to other molecular biology techniques is:

A. low risk of contamination

B. rapidity

C. low sensitivity

D. low specificity

 

Answer:  B. rapidity. PCR allows the creation of billions of copies of a specific DNA fragment or gene, which allows for detection and identification of gene sequences using visual techniques based on size and charge. It is a highly sensitive technique and can allow results within a shorter time frame than most molecular techniques.

 

 

4. The PCR process contains these three steps:

A. denaturation, transcription, annealing

B. annealing, denaturation, transcription

C. denaturation, annealing, transcription

D. transcription, annealing, denaturation

 

Answer:  C. denaturation, annealing, transcription. The PCR process can be divided into three main steps. The denaturation process allows the solution to be heated above the melting point of the two complementary strands of the template DNA, which allows the strands to separate. Then the annealing process allows the primers to bind to the specific DNA segment. The annealing between the primers and the template DNA occurs only if they are complementary in sequence. The temperature is raised again at which time the DNA polymerase is able to extend the primers by adding nucleotides to the developing DNA strand, known as transcription. With each repeat of these three steps, the number of copied DNA molecules is doubled.

Research Techniques Made Simple: COMPARATIVE EFFECTIVENESS RESEARCH

For details, we refer you to the article “Research Techniques Made Simple:  COMPARATIVE EFFECTIVENESS RESEARCH” by Vinod E Nambudiri and Abrar Qureshi at http://www.jidonline.org.

 

Questions

1.  Study designs used for comparative effectiveness research include:

a) Systematic review

b) Randomized controlled trial

c) Cross sectional study

d) All of the above

 

2. Differences in survival between two treatment groups are best compared using which of the following statistical methods?

a) Paired t-test

b) Log-rank test

c) ANOVA

d) Fisher’s exact test

 

3. The intraclass correlation coefficent representing the best degree of agreement between two diagnostic tools among the following is:

a) ICC < 0.01

b) ICC = 0.05

c) ICC = 0.50

d) ICC = 0.95

 

____________________________________

 

Answers:

 

1.  Study designs used for comparative effectiveness research include:

d) All of the above

 

2. Differences in survival between two treatment groups are best compared using which of the following statistical methods?

               b) Log-rank test

 

3. The intraclass correlation coefficent representing the best degree of agreement between two diagnostic tools among the following is:

               d) ICC = 0.95

 

Research Techniques Made Simple: IMMUNOFLUORESCENCE Q&A

For details, we refer you to the article “Research Techniques Made Simple: Immunofluorescence Techniques” by Ian D. Odell1, MD, PhD and Deborah Cook2, MD

Departments of Medicine1 and Pathology2 University of Vermont College of Medicine, Burlington, VT

Questions

  1. What is the purpose of Michel’s solution?
    1. Fix the tissue before detection
    2. Cross link cellular components to retain the integrity of cellular structures
    3. Disrupt plasma membranes to give antibodies access to their target antigens
    4. Precipitate the immune complexes to preserve antigenicity

 

  1. How many additional antibodies are required to detect autoimmune complexes?
    1. 1
    2. 2
    3. 3
    4. 4

 

  1. Which of the following techniques is more sensitive than immunofluorescence for the diagnosis of some autoimmune bullous diseases?
    1. Light microscopy
    2. ELISA
    3. Dermatoscope
    4. Western blot

Answers

1. D

2. A

3. B

RESEARCH TECHNIQUES MADE SIMPLE: CONFOCAL MICROSCOPY Q&A

These questions and answers relate the the article “RESEARCH TECHNIQUES MADE SIMPLE:  INTRODUCTION TO CONFOCAL MICROSCOPY” by Nwaneshiudu et al.  For details see the December 2012 issue of JID.

 

 

QUESTIONS

 

1) Features of confocal microscopy include which of the following:

a. Formation of the focal point of the objective lens on a pinhole to decrease “noise”

b. Increase in the optical resolution and contrast of the image

c. Ability to reconstruct a 3-D image of the specimen

d. Ability to collect serial optical sections from thick specimens

e. All of the above

 

 

2) Which microscope uses a series of moving pinholes on a disk?

a. Programmable array microscope

b. Spinning-disk confocal microscope

c. Scanning transmission electron microscope

d. Phase-contrast microscope

 

 

3)  What is the role of a photomultiplier tube?

a. It collects fluorescence at the dichroic mirror
b. It provides the excitation light

c. It scans the emitted light
d. It detects the emitted light

 

4)  What may be the consequence of using two different fluorescent dyes?

a. Photobleaching

b. Phototoxicity

c. Chromatic and spherical aberration
d. Less detectable photons

 

ANSWERS

1) Features of confocal microscopy include which of the following:

e:  All of the above.

 

 

2) Which microscope uses a series of moving pinholes on a disk?

b:  Spinning-disk confocal microscope

 

3)  What is the role of a photomultiplier tube?

d:  It detects the emitted light

 

4)  What may be the consequence of using two different fluorescent dyes?

c:  Chromatic and spherical aberration