methods 5 Flashcards
What technology, RT-PCR or qRT-PCR, will you use to detect alternative splicing (presence versus absence of an intron)? How will you design your primers (i.e., where would they anneal with respect to the alternatively spliced intron)?
To detect alternative splicing (is intron retained or spliced out?) rt-pcr is fine, just need a yes or no answer of does it amplify and rt-PCR is cheaper and simpler
To design primers: since you’re running it on a gel you will be able to tell size difference of fragments. One primer should bridge the gap of an exon (in an are up/downstream of GOI, just to ensure to gDNA contaminanation), the other primer should be outside of the exon. When you run on a gel the spliced and unspliced fragments will run as different sizes
As rtPCR is semiquantative you’d follow up with q-rt-PCR is you want to compare expression, you would have to run 2 reaction per sample (since you wont be sorting by size)
What method, ddPCR or nanostrings, will you use to compare the expression of two genes of interest in two samples? How about 100 genes in two samples? How about two genes in 100 samples? And 100 genes in 100 samples?
two gene: ddPCR
100 genes 2 samples: nanostring (1 reaction for all genes)
2 genes 100 samples: ddPCR
100 genes 100 samples: nanostrings
what does a microarray measure
comparative gene expression between two samples/treatments
what is on a microarray chip?
droplets of DNA attached to the chip (spotted on or PCR performed on chip)
example of microarray method protocol?
glass slide is spotted with DNA that was PCR amplified
RNA is extracted from 2 samples you want to compare
RNA –> cDNA, tagged with green/red fluorescence
hybridized to slide, by color you can tell which samples expressed higher levels of genes on array by color
explain Genechip oligonucleotide arrays (what is measured/how are samples prepped)
presence/absence of relative levels of thousands of genes in a sample, 100s of genes
total mRNA rt-PCR with oligo-dt, then in vitro trancription back to RNA and incorporate Biotin. now you have biotin labeled cRNA.
labeled cRNA hybridized to the microarray, washed, stained, quantified
how to use microarrays for genotyping?
You can’t sequence whole genome, but you can screen for variants of a gene
A chip has oligos representing different allelic versions of gene of interest
Fragmented DNA is end-labeled with fluorescent dye and hybridizing
fluorescence read, spots that light up are interpreted, is multiple spots light up the heterozygous for a polymorphisms, only one spot = one allele present
what is the purpose of a tiling array?
can design oligo probes to overlap and cover as many genes as you want
examples of how to use microarray for transcriptome analysis
gene discovery
gene expression
alternative splicing
RNA-binding protein transcript target id
examples of how microarrays can be used for genome analysis
ChIP-chip
methylome analysis
genome resequencing
polymorphism discovery
comparative genome hybridization
How can NGS be applied to study gene expression and what needs to be done to prepare RNA samples for NGS?
RNA has to first be converted to cDNA, then you can use tech like illumina to sequence it
Is this a good approach for looking at expression of a single gene of interest across multiple conditions (e.g., in different tissues, at different stages of development, under different stresses, in different genotypes/lines/mutants, etc.)?
multiple genes across limited samples
because you are looking at the whole genome worth of genes it can get complicated comparing too many samples
What types of additional information would RNA-seq provide that other methods of gene expression analysis could not?
alternative splicing (microarrays could only tell you this with a tiling array)
what makes it easier to align reads after RNA sequencing?
a reference genome, even rare reads can be aligned
what is IGV
integrative genome viewer, aligns RNA reads
purpose of SDS-PAGE
measure protein expression
what is the purpose of SDS in SDS-PAGE
coat the proteins with a uniform negative charge, the charge impacts the speed that protein runs on gel so if you want to compare sizes you need to have negative charge
what is purpose of b mercaptoethanol is SDS-PAGE
reducing agent to break disulfide bridges in proteins, will break molecular interactions
why do you boil samples in SDS-PAGE
to denature the proteins
what type of reaction allows visual detection of protein of interest in western blot
chemiluminescent reaction
purpose of blocking step
block sticky proteins from binding up antibody, lower binding affinity
Northern blots are to _____ as western blots are to _____
in situ hybridization
immunolocalization
immunolocalization measures what
protein/biomolecules in tissue, cell, of lysate
how does immunolocalization work?
antibody based, similar principle to western blot, tagged with fluorophore, enzyme, or gold particles
affinity purification of proteins is what?
a chromatographic technique for the isolation of protein of interest from complex samples
a way to purify your protein of interest
Use recombinant organism to express tagged GOI, resin in column is cross-linked to ligand, protein will bind to the column and others will be washed our
why may you end up with multiple bands on a gel after running product from affinity purification?
degredation products of your protein or a sticky protein may bind column
what does yeast-2 hybrid measure
interaction between 2 proteins of interest
explain yeast 2 hybrid methods
two hybrid proteins:
bait = DNA binding domain + target protein
prey = transcriptional activation domain + binding partner
recombinant genes encoding bait and prey are introduced into yeast cell, bait binds DNA, if binding partner binds target the transcriptional activation domain will activate reporter gene transcription
what types of reporter can you use in yeast 2 hybrid?
auxotrophic or colorimetric
how can a yeast 2 hybrid be used to identify novel interactors of protein of interest?
you want to know how your protein of interest interacts with the entire genome worth of proteins, start with a cDNA library from your sample (cDNA is transformed into many yeast so you have a variety of yeast all expressing different proteins), you have yeast now that expresses both your protein of interest (bait) and one of the genes expressed (prey) the colonies which survive/ turn blue are colonies that had cDNA belonging to an interection partner of you protein, collect and sequence them
