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
