In Vitro Experimentation Flashcards
How do you isolate DNA, RNA and proteins - solution based
Starting material - be liquid, cells in suspension, homogenised tissue, blood components e.g. buffy coat
These are lysed, precipitated and separated into supernatant and the protein precipitate
The protein precipitate can undergo treatment to leave RNA, DNA or proteins/lipids
RNA = aqueous phase
Interphase = DNA
Organic phase = proteins, lipids
How do you isolate DNA, RNA and proteins - column based
Columns are inserted into a tube and has different affinities
Solution is passed through the column and the target molecule binds to the column
DNA = binds to allprep column, column washed, then DNA eluted
RNA = DNA binds allprep, washed out and into RNeasy column, column washed, RNA eluted
Protein = DNA binds allprep, washed out and into RNeasy column, washed out into new tube and precipitated to form protein
How can you measure DNA, RNA and protein quality/quantity
Spectrophotometry
DNA and RNA - 260nm, Protein =280nm
The more light absorbed, the higher the concentration = high OD
Quality
260/280 - pure RNA/DNA = 2.1/1.8, lower ratio = protein contamination
260/230 - pure =1.8, lower ratio - organic contaminants
Absorbance peak = smooth peak shows no contamination
How do you isolate DNA, RNA and proteins - column based
Columns are inserted into a tube and has different affinities
Solution is passed through the column and the target molecule binds to the column
DNA = binds to allprep column, column washed, then DNA eluted
RNA = DNA binds allprep, washed out and into RNeasy column, column washed, RNA eluted
Protein = DNA binds allprep, washed out and into RNeasy column, washed out into new tube and precipitated to form protein
What methods can be used to analyse DNA
Gel electrophoresis - higher% = smaller fragments = higher resolution
PCR - requires MgCl2, buffer, dNTP, template, primer and polymerase - denature, anneal extension repeat
Sanger sequencing - PCR with fluorescent chain terminators
NGS - DNA extraction, shearing, ligation, library construction, amplification, sequencing, assembly, bioinformatics
What is the effect of mutations in noncoding DNA
Regulatory elements (e.g. TATA box) - affects level of gene expression
Spicing of introns - highly conserved GT and AG at the end of introns
Coding sequences lost, or introns kept in mRNA
What methods can be used to analyse RNA (differential gene expression analysis)
qRT-PCR - for gene expression analysis, generate cDNA from mRNA followed by PCR, compared to standard curve graph
NMD assay (nonsense mediated mRNA decay) - for mRNA stability
Minigene assay - for splicing variants analysis
Also - Micro-array, and RNA-seq
How is qRT-PCR performed
For proteins - reverse transcription to transcribe mRNA into cDNA:
RNA-dependent DNA polymerase assembles cDNA from RNA template
RNase H degrades the RNA template
DNA-dependent DNA polymerase creates the second DNA strand
qPCR:
Uses dNTP labelled so that it fluoresces when bound - output measured real time in thermal cycler
Number of cycles taken to reach threshold value is determined as it is proportional to the amount of starting cDNA (mRNA)
What is nonsense-mediated mRNA decay
If mRNA is not found in a sample might be due to NMD
Exon-junction complexes sit at the interaction between exons and intron, marking splice sites
When the ribosome transcribes the mRNA, it will displace the EJC upon encounter
If a premature stop codon is present, the EJC is retained, inducing NMD
NMD occurs via a signalling cascade linked to the EJC which then degrades the RNA
How do you prevent NMD/ check if it is occurring
Cycloheximide (eukaryote protein synthesis inhibitor) interferes with NMD through inhibition of protein synthesis and accumulation of mutated mRNA
This is used to view if NMD is occurring indicating a premature stop codon
NMD activity in a particular model can be evaluated by classical RNA quantification methods e.g. qPCR
How is the minigene assay performed
They assess the impact of sequence variants on splicing
A genomic segment containing the variant sequence of interest along with flanking intronic sequences is amplified via PCR and cloned into a minigene vector
After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the variant constructs are compared by reverse transcription-PCR analysis and sequencing/gel electrophoresis
This method represents a complementary approach to reverse transcription-PCR analyses of patient RNA for the identification of pathogenic splicing mechanisms
What methods are used to analyse proteins
SDS-PAGE - separation of proteins based on molecular weights
Western blot - detection of proteins using specific antibodies
How is SDS-PAGE performed
SDS- based solution, Acrylamide gel
Loading dye (density-e.g. glycerol- plus colour-e.g. bromophenol blue), prevents protein floating, and allows visualisation
Visualised by Coomassie blue stain
Proteins negatively charged due to SDS move to a positive electrode
They are separated by size (molecular weight) -smaller proteins move faster
How is western blot performed
Blot paper sandwiches a membrane and gel electrophoresis is run
The membrane is then taken to be incubated with the antibody solution
Isolate all proteins from sample → Gel electrophoresis → Transfer to membrane → Block → ELISA; Primary antibody → Secondary antibody
ELISA Immunofluorescence - The primary antibody is designed to bind to the protein of interest, the secondary antibody is designed to bind the IgG
Colorimetric - attached to enzyme, which causes a substrate colour change
Chemiluminescence - attached to enzyme, formation of product releases light, printed on X-ray film
Fluorescence - conjugated with fluorophore which emits light
Describe different forms of alternate splicing
Exon skipping or cassette exon - an exon may be spliced out of the primary transcript or retained
Alternative donor site - an alternative 5’ splice junction (donor site) is used
Alternative acceptor site - an alternative 3’ splice junction (acceptor site) is used
Intron retention - a sequence may be spliced out as an intron or simply retained
Mutually exclusive exons - one of two exons is retained in mRNA after splicing but not both
What is immunohistochemistry
Cells within a tissue can undergo a similar process as immunofluorescence, however the secondary antibody is conjugated to an enzyme (colorimetric)
This enzyme will react with reagents to create a coloured precipitate where the antibody is bound e.g. horseradish peroxidase
This is seen under a microscope
Describe multiplex protein detection - protein assays
Membranes are immobilised with different antibodies and then incubated
There would be two versions - treated and untreated antibody-protein complexes? which are compared
You can then see how a protein is affected by a treatment
How do you analyse protein-protein interactions
Immunoprecipitation and Co-IP
This allows you to analyse protein complexes from a mixture by isolating them via an antibody that binds one of the proteins in a complex
An antibody is added to the mixture, followed by a protein A or G coupled bead with an affinity for the antibody
The tube is spun and all the antibodies bound to the beads along with the complex fall to the bottom as an ‘immunoprecipitated’
What is Mass Spec and what is it used for
Mass spectrometry is a sensitive technique used to detect, identify and quantitate molecules based on their mass-to-charge (m/z) ratio, in this case proteins
Proteins are extracted from cells/tissues, and digested into peptides
Peptides are attached to support and a laser ionises them and are accelerated through drift tube
Time Of Flight measured– correlates with mass of molecule
Ionization allows scientists to obtain protein mass “fingerprints” that could be matched to proteins and peptides in databases and help identity unknown targets
How do you analyse DNA-protein interactions plus give an example of DNA-protein interactions
E.g. Transcription factors
Luciferase reporter assay
EMSA - electrophoretic mobility shift assay
ChIP-Seq
Describe EMSA - Electrophoretic Mobility Shift Assay
Determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence
Based on gel electrophoresis
Identifies changes in mobility of fragments
Requires protein, probe, antibody and specific competitor and mutant/non competitor
Lane 1 = 1 probe
Lane 2 = protein + probe - shift is shown as a band is formed somewhere else
Lane 3/4 = protein + competitors - shows if binding is specific
Lane 5 = protein + probe + antibody - super shift proves if it is actually the protein of interest
Describe ChIP-Seq to identify TF binding sites (or other protein-DNA interaction)
Chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing
• Steps ○ Transcription factors bound to DNA in nucleus ○ Fix the protein to the DNA, then shear ○ Immunoprecipitate TF of interest, along with attached DNA with Ab complementary to protein Sequence the DNA to determine the binding site
Describe the use of luciferase for TF DNA-RNA interactions
TFs are an example of DNA-protein interaction
Luciferase reporter assay for transcription factor activity
Requires a plasmid which expresses the luciferase enzyme when TFs bind its promoter
The reaction of the enzymes releases light
If light is seen, it means TFs bound its promoter to induce transcription
What are the cell types used in in vitro techniques
Primary cells
Cell lines
Stem cells
