Experimental Methods Flashcards
You are designing an experiment. You are investigating the presence of miRNA AB7456. Which if the following is the MOST appropriate?
a. Southern blot
b. Northern blot
c. Western blot
d. mass spectrometry
e. flow cytometry
B. Northern
A cell line is being prepared from a patient’s tissues. The point at which this cell line becomes senescent is called:
a. telomerase finality
b. Pautrier’s cessation
c. Hayflick limit
d. primordial finality
e. limiting expansion
C - Hayflick
You plan to investigate lymphoma cell lines after exposure with azacitidine, a DNA methyltransferase inhibitor. Which of the following would be most useful to analyze methylation of the cell’s DNA?
a. sodium bisulfite conversion
b. RT-PCR
c. Southern blotting
d. flow cytometry
e. mass spectrometry
A - sodium bisulfite conversion
page 23
Which of the following techniques is suitable for identifying protein within a sample?
a. Western blotting
b. Southern blotting
c. Northern blotting
d. Eastern blotting
A - Western
T&H, page 8. Northern blotting is used to characterize mRNA. Southern blotting = DNA, Western blotting = proteins, Eastern blotting = protein post-translational modifications.
You are performing a DNA microarray experiment using cDNA extracted from 13 canine mammary carcinomas. Your gene of interest fluoresces red within the majority of the samples. What is your conclusion?
a. This gene is hypermethylated in the tumor cells compared to controls.
b. Gene expression is down-regulated in the tumor population compared to the reference sample.
c. Gene expression is up-regulated in the tumor population compared to the reference sample.
d. There is no change in gene expression.
e. This gene is deleted from the tumor population, though expressed in normal tissues.
C. gene is upregulated in tumor
Explanation: T&H, page 20-21. Microarray analysis uses cDNA from your sample, which is attached to a red fluorescent label. cDNA from normal tissue (reference) is attached to a red fluorescent label. These samples are hybridized to a microarray that contains single-stranded DNA. Blocks in which the normal tissue gene is expressed will fluoresce red, those where the tumor gene is expressed will fluoresce green, and those where both are expressed will fluoresce yellow.
- Which of the following is represented by the p-value?
i. Type I error
ii. Type II error
iii. β error
iv. The probability of false-positives
v. The probability of false-negatives
a. ii, iii, v
b. i, iii, iv
c. i, iv
d. ii, iii, iv
e. i, v
C. Type I, prob of false pos
Explanation: T&H, page 44. The p-value represents Type I or α error. This also reflects the probability of false-positive results. Type II (or β error) is the failure to reject a false null hypothesis (a false negative).
Which of the following is the standard technique used to determine known cytogenetic aberrations (amplifications, deletions, whole chromosome abnormalities) in certain tumor types using tumor biopsies or cultured cells. Examples include: HER2 status of breast cancer and N-myc amplification in neuroblastoma.
a. Comparative genomic hybridization
b. Polymerase chain reaction
c. Fluorescence in situ hybridization
d. Cyclic reversible termination
c - FISH
Explanation: FISH can be performed on interphase nuclei from paraffin embedded tumor biopsies or cultured tumor cells, which allows cytogenic abberations such as amplifications, deletions, or other abnormalities of whole chromosomes to be visualized without the need for obtaining good quality metaphase preparations. This technique is also useful for the detection of the bcr-abl rearrangement in CML and the tmprss2-erg in prostate cancer.
Identify the most correct statement in regards to statistical analysis.
a. The statistical power of analysis is called a Type I or α error and indicates that a false-positive result will be avoided
b. The analysis of survival is usually performed using t-tests
c. The Cox-proportional hazards model enables the difference between survival times or particular groups of patients to be tested while allowing for covariates, each of which is likely to influence survival
d. The p-value represents the chance of making a Type II (or β) error (a false-negative) for testing a single hypothesis
C - Cox prop. hazards
a. The statistical power of analysis is called a Type II or β error and indicates that a false-negative result will be avoided
b. The analysis of survival is usually performed using log-rank analysis
d. The p-value represents the chance of making a Type I (or α) error (a false-positive) for testing a single hypothesis
What is a limitation of comparative genomic hybridization?
a. it can detect the presence of genetic aberrations, but not their chromosomal location
b. it can only detect alterations that result in a copy number change or translocation of a DNA segment
c. it requires prior knowledge of the approximate location of the chromosomal abnormality
d. the radioactive probes involved require specific safety/handling considerations
b. Can only detect copy number change or segment translocation
Explanation: see T&H p. 11. (a) Hybridization to a normal metaphase chromosomal spread helps determine location. (c) the benefit of CGH is it does NOT require prior knowledge of the aberration’s location (or even its existence). (d) CGH typically uses fluorescent rather than radioactive probes.
Which of the following is/are true regarding microRNAs (miRNA)?
i. they evolved as protective strategies against viral infection
ii. formalin fixation destroys its structural integrity for later analysis
iii. they are primarily derived from introns and other non-coding sequences
iv. their activity is antagonized by RISC (RNA-induced silencing complex)
v. most miRNA undergoes extensive post-transcriptional processing before reaching full activity
a. ii, iii, v
b. i, ii, iv
c. ii, iv, v
d. i, iii, v
d.
Explanation: pp. 26 and 40, T&H. (ii) formalin fixation has little effect on miRNA structural integrity. (iv) miRNA is incorporated into RISC, not antagonized by it
Which of the following proteins would require an extra preparatory step for the sample prior to being analyzed via flow cytometry?
a. FoxP3
b. MHCII
c. P-glycoprotein
d. CD4
a. FoxP3
Explanation: All of the others are cell surface proteins that can be accessed directly via antibody. FoxP3, being a transcription factor, is located intracellularly and requires a permeabilization step to allow antibody access.
Which of the following statement is true regarding epigenetics and cancer?
a. Epigenetics relates to heritable changes in gene expression
b. Epigenetic changes are encoded in the genome
c. In general cancer cells exhibit genome wide hypermethylation
d. Methylation of DNA promotes DNA polymerase binding
a. Epigenetics relates to heritable changes in gene expression
Explanation: T&H, pages 20-22. Epigenetic changes are NOT encoded in the genome, but are heritable. Cancer cells generally exhibit genome wide hypomethylation with local hypermethylation of CpG islands associated with promoters. DNA methylation causes gene silencing.
Which of the following best describes microRNA and its function within eukaryotic cells?
a. MicroRNA is transcribed by RNA polymerase from the non-sense strand of DNA
b. It functions to upregulate gene expression by binding to the N-terminal tail
c. MicroRNA participates in RNA silencing
d. MicroRNA represents genetic material that can be easily transfected from one cell to another
c - MicroRNA participates in RNA silencing
Explanation: T&H; page 26.
Which of the following tests is matched with the appropriate target for analysis?
a. Cytogenic aberrations – mass spectrometry
b. Single nucleotide polymorphisms – fluorescence in situ hybridization
c. DNA methylation – northern blotting
d. Chromosomal translocation – comparative genomic hybridization
d: Chromosomal translocation – comparative genomic hybridization
Explanation: T&H Ch2
- Mass spectrometry – mass, structure, and elemental composition of molecule (i.e. protein)
- SNPs – DNA sequencing
- FISH – Cytogenetic aberrations; gene identification on chromosomes or whole chromosome abnormalities
- DNA methylation – sodium bisulfate conversion
- Gene expression patterns using RNA – northern blotting
