Single Molecule Genomics Flashcards
Why count single(RNA) molecules?
- Sensitivity -> the expression of this cell is three times the amount of it’s mother
- Accuracy–>high accuracy if a method is very sensitivity
- subcellular localization
- RNA targetable by sequence
- Absolute quantification
Absolute quanitification
- Allow labs to compare results directly
- very useful for computational modeling
- allows numerical inference
Methods for single molecule quantification of RNA
PCR-based methods
- single cell RT-PCR
- Digital RT-PCR
Imaging-based methods
- In-situ hybridization
- direct detection
- signal ampliflication
Traditional qRT-PCR
averages many cells
meat grinder (lose spatial info)
then perform PCR
the earlier the pcr
pcr reaction vs time based on number of cycles
the earlier product reaches a threshold the more starting material there was in the cell
qRT-PCR:
How to obtain single cells?
–cells–>isolate–>dilution
-manual dissection/aspiration
-laser capture
-collection of cell use a specific resin and hit with a laser to pick up cells and purify them
-flow cytometry
bunch of cells going throw a tube and use a laser to identify characteristics, and have it go through certain tubes
RNA-prep (some lost of molecules)
qRT-pcr
lyse
reverse transcribe
qPCR amplify
Advantages of single cell RT-PCR
- cheap
- high dynamic range
- can detect SNPs and distinguish mRNA isoforms
- relatively easy to multiplex many genes(if abundant enough)
Cons of single cell RT-pcr
difficult to calibrate for absolute measurements
sensitivity not 100%(careful analyses indicate ~10 RNA per cell is the limit)
Digital RT-PCR
solves calibration problem
- cell contents split into hundreds or more individual PCR reactions
- each run gives on and off measurement of the presence or absence of the target RNA
Digital RT-PCR cons
- reduced dynamic range
- complex microfluidics or emulsions
- harder to multiplex
- not efficient to do large number sizes
Imaging-based methods
fixed methods
-fluorescence in situ hybridization (FISH)) and can target endogenous RNAs
-live cell methods
RNA molecule into cell
(engineered RNAs) *hybridization or GFP based
let’s you look at movement over a period of time
Imaging-based: Fixed cells
Direct detection
Amplified or indirect detection
Direction detection
is more accurate and simple but requires somewhat more complex microscopy and cannot detect SNPs and microRNA
possible with multiple/tiled oligonucleotides
(5-10 oligo probes, 50 bases each, multiple labeled)
30-60 oligo probes, 20 bases each, singly labeled (Raj)
Amplifies detection
Has a higher rate of false positives and negatives but can detect shorter nucleic acids and different isoforms
Bursting
switching between on and off states
mRNA follows a Poisson distribution if no bursting, increased variability if bursting
Signal amplification
produces ore singal per molcule
various schemes generate a lot of fluorescence from a single probe
often LNA to increase specificity; can be used for snp and microRNA detection
rate of false negative and positive is still unclear
LNA probe ??
locked nucleic acid, ink at the back that locks in for base pairing and stacking
Nuleic acid amplification schemes
Branched DNAs give more targets to probes
Rolling circle amplification of padlock probes uses a nucleic acid amplification followed by direct FISH detection( this can detect SNPs)
Live imaging
Two methods: MS2 and molecule beacons
MS2 method
(GFP based)
requires genetically engineered transonic ran with special sequence tag
Molecular beacons
alternate method for visualizing individual transcripts in live cells
closed until it hybridizes with mRNA and then it fluroresses
allows you to watch transcriptional burst in real time
Important features of biology revealed by single molecule
- transcription happens in bursts
- ms2 reveal characteristics of motion through nuclear pore
- movement of mRNAS explained by random brownian motion
Downsides of MS/Beacons
bother require engineered mRNAS
MS2 suffers from clumping artifacts and changes mRNA half-life in some cases
Molecular beacons are hard to deliver to cells and may have unknown effects upon the mRNA dynamics.
oligonucleotides are subject to various unknown cellular processes
visualizing single protein molecules
much more challenging-can’t tile probes like for mRNA
- diffusion limits time averaging for most molecules
- requires very sensitive microscope like TURF
fluorescent protein detection
1) use brighter fluor
2) sensitive microscope methods(TIRF)
3) Time inebriation can help, if applicable
gfp imaging
allows detecting stochastic decision making thresholds
optical trapping
powerful in vitro exist for single molecule anyalsis
beads under light that keep them immobilize, tether ran polmerase and dna on the other
measure individual steps between
long time - nucleotide
biophysical study to look at how motors work
DNA curtains
one end attachment areas
dna stuck to those anchors
flow or electric current cause DNA to stretch our and you get these stretch out arrays of DNA, stain them
Helicos-single molecule methods
direct imaging of fluorescent reversible terminators in single elongating DNA molecules
Pacific Bioscience-single molecule methods
Residence time of flurorescently labeled dNTPs at immobilized polymerase
Oxford nanopore-single molecule methods
pass single molecules through pore and measure electrical signal
