Single Molecule Genomics Flashcards

1
Q

Why count single(RNA) molecules?

A
  • 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
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2
Q

Absolute quanitification

A
  • Allow labs to compare results directly
  • very useful for computational modeling
  • allows numerical inference
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3
Q

Methods for single molecule quantification of RNA

A

PCR-based methods

  • single cell RT-PCR
  • Digital RT-PCR

Imaging-based methods

  • In-situ hybridization
    • direct detection
    • signal ampliflication
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4
Q

Traditional qRT-PCR

A

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

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5
Q

qRT-PCR:

How to obtain single cells?

A

–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)

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6
Q

qRT-pcr

A

lyse
reverse transcribe
qPCR amplify

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7
Q

Advantages of single cell RT-PCR

A
  • cheap
  • high dynamic range
  • can detect SNPs and distinguish mRNA isoforms
  • relatively easy to multiplex many genes(if abundant enough)
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8
Q

Cons of single cell RT-pcr

A

difficult to calibrate for absolute measurements

sensitivity not 100%(careful analyses indicate ~10 RNA per cell is the limit)

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9
Q

Digital RT-PCR

A

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
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10
Q

Digital RT-PCR cons

A
  • reduced dynamic range
  • complex microfluidics or emulsions
  • harder to multiplex
  • not efficient to do large number sizes
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11
Q

Imaging-based methods

A

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

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12
Q

Imaging-based: Fixed cells

A

Direct detection

Amplified or indirect detection

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13
Q

Direction detection

A

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)

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14
Q

Amplifies detection

A

Has a higher rate of false positives and negatives but can detect shorter nucleic acids and different isoforms

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15
Q

Bursting

A

switching between on and off states

mRNA follows a Poisson distribution if no bursting, increased variability if bursting

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16
Q

Signal amplification

A

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

17
Q

LNA probe ??

A

locked nucleic acid, ink at the back that locks in for base pairing and stacking

18
Q

Nuleic acid amplification schemes

A

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)

19
Q

Live imaging

A

Two methods: MS2 and molecule beacons

20
Q

MS2 method

A

(GFP based)

requires genetically engineered transonic ran with special sequence tag

21
Q

Molecular beacons

A

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

22
Q

Important features of biology revealed by single molecule

A
  • transcription happens in bursts
  • ms2 reveal characteristics of motion through nuclear pore
  • movement of mRNAS explained by random brownian motion
23
Q

Downsides of MS/Beacons

A

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

24
Q

visualizing single protein molecules

A

much more challenging-can’t tile probes like for mRNA

  • diffusion limits time averaging for most molecules
  • requires very sensitive microscope like TURF
25
fluorescent protein detection
1) use brighter fluor 2) sensitive microscope methods(TIRF) 3) Time inebriation can help, if applicable
26
gfp imaging
allows detecting stochastic decision making thresholds
27
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
28
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
29
Helicos-single molecule methods
direct imaging of fluorescent reversible terminators in single elongating DNA molecules
30
Pacific Bioscience-single molecule methods
Residence time of flurorescently labeled dNTPs at immobilized polymerase
31
Oxford nanopore-single molecule methods
pass single molecules through pore and measure electrical signal