Experimental Methods Flashcards
RNA levels can be determined by:
- Microarray and RNAseq (transcriptomic approach)
- Northern Blot and qPCR (gene-specific
RT-qPCR
- A method used to quantify the level of a specific RNA in cell
PCR (Polymerase Chain Reaction)
- A method widely used to rapidly make millions to billion of copies of a specific DNA sample
qPCR
Quantitative PCR
- Moniters the amplification of a targeted DNA molecule during the PCR in real-time.
RT-qPCR
Reverse Transcription to generate cDNA followed by quantitative PCR using real time PCR machine to determine RNA levels.
Types of Evidence
- Correlative
- Loss-of-function
- Gain-of-function
Gain-of-function (methods)
- 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.
Loss-of-function (methods)
- Delete gene from the chromosome
- Reduce expression using RNAi (RNA interference)
- Reduce activity using inhibitors
Plasmid
Small, usually circular, extrachromosomal DNA molecule within a cell that
can replicate independently.
Found commonly in bacteria, but are also present in some eukaryotes.
Expression Vectors
are those designed for the expression of a gene in cells
RNAi: RNA interference
A biological process in which double-stranded RNA molecules (siRNA or miRNA) inhibit gene expression or translation by neutralizing targeted RNA molecules.
siRNA: small interfereing RNAs
Usually complementary RNAs that come from (1) endogenous sequences that are not genes or (2) exogenous sources such as those produced by RNA viruses
shRNA: Short hairpin RNA
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.
Cell Proliferation Assay
- MTT assay
- Colony formation assay
- Cell cycle analysis using flow cytometry
MTT assay
Used to measure cellular metabolic activity as an indicator of cell
viability, proliferation and cytotoxicity.
(makes sure cells are proliferating)
Ubiquitin
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.
Deubiquitinase (DUB)
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
Proteasome
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
K-48 isopeptide linkage
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.
Ubiquitin has…
7 lysines
- ubiquitin chains can have a distinct structure depending on which (K) residue within ubiquitin is utilized to form the chain
Ubiquitin-like proteins (UBLs)
● 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
