Experimental Methods

Question 1

RNA levels can be determined by:

Answer 1

• Microarray and RNAseq (transcriptomic approach)
• Northern Blot and qPCR (gene-specific

Question 2

RT-qPCR

Answer 2

• A method used to quantify the level of a specific RNA in cell

Question 3

PCR (Polymerase Chain Reaction)

Answer 3

• A method widely used to rapidly make millions to billion of copies of a specific DNA sample

Question 4

qPCR

Answer 4

Quantitative PCR
- Moniters the amplification of a targeted DNA molecule during the PCR in real-time.

Question 5

RT-qPCR

Answer 5

Reverse Transcription to generate cDNA followed by quantitative PCR using real time PCR machine to determine RNA levels.

Question 6

Types of Evidence

Answer 6

• Correlative
• Loss-of-function
• Gain-of-function

Question 7

Gain-of-function (methods)

Answer 7

• Transfect cells with a plasmid that will express (over-express) the gene of interest.
• Introduce the molecule of interest itself (typically an RNA) into the cell so that its levels are substantially higher.

Question 8

Loss-of-function (methods)

Answer 8

• Delete gene from the chromosome
• Reduce expression using RNAi (RNA interference)
• Reduce activity using inhibitors

Question 9

Plasmid

Answer 9

Small, usually circular, extrachromosomal DNA molecule within a cell that
can replicate independently.
Found commonly in bacteria, but are also present in some eukaryotes.

Question 10

Expression Vectors

Answer 10

are those designed for the expression of a gene in cells

Question 11

RNAi: RNA interference

Answer 11

A biological process in which double-stranded RNA molecules (siRNA or miRNA) inhibit gene expression or translation by neutralizing targeted RNA molecules.

Question 12

siRNA: small interfereing RNAs

Answer 12

Usually complementary RNAs that come from (1) endogenous sequences that are not genes or (2) exogenous sources such as those produced by RNA viruses

Question 13

shRNA: Short hairpin RNA

Answer 13

An artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNAi. Expression of shRNA in cells is typically accomplished by delivery of plasmids or through other vectors.

Question 14

Cell Proliferation Assay

Answer 14

1. MTT assay
2. Colony formation assay
3. Cell cycle analysis using flow cytometry

Question 15

MTT assay

Answer 15

Used to measure cellular metabolic activity as an indicator of cell
viability, proliferation and cytotoxicity.
(makes sure cells are proliferating)

Question 16

Ubiquitin

Answer 16

Small [76 amino acid], highly conserved protein present in all eukaryotic cells that can be covalently attached to lysines of other proteins. Attachment of a short chain of ubiquitins to such a lysine can tag a protein for intracellular proteolytic destruction by a proteasome.

Question 17

Deubiquitinase (DUB)

Answer 17

Cleaves ubiquitin from proteins including (but not limited to):
● Ubiquitin-tagged proteins
● Ubiquitin units in a free polyubiquitin chain
DUBs can reverse ubiquitination and recycle ubiquitin by cleaving
ubiquitin chains

Question 18

Proteasome

Answer 18

Large protein complex in the cytosol with proteolytic activity that is responsible for degrading proteins that have been marked for destruction by ubiquitylation or by some other means

Question 19

K-48 isopeptide linkage

Answer 19

the primary signal for proteasome dependent degradation of proteins. The attachment of a chain of four or more ubiquitin molecules to a protein is required for efficient degradation.

Question 20

Ubiquitin has…

Answer 20

7 lysines
- ubiquitin chains can have a distinct structure depending on which (K) residue within ubiquitin is utilized to form the chain

Question 21

Ubiquitin-like proteins (UBLs)

Answer 21

● A family of small, non-enzymatic proteins that share a common structure
and are involved in post-translational modification of other proteins
● Usually have a regulatory function
- - - Ubiquitin chains can have a distinct structure depending on which Lys (K) residue within ubiquitin is utilized to form the chain
● The human genome encodes at least eight families of UBLs including SUMO, NEDD8, ATG8, and ATG12
● The different UBLs have parallel mechanisms for their covalent conjugation to target proteins