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Flashcards in Single Molecule Genomics Deck (31)
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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
2
Q

Absolute quanitification

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

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)

6
Q

qRT-pcr

A

lyse
reverse transcribe
qPCR amplify

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)
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)

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
10
Q

Digital RT-PCR cons

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

12
Q

Imaging-based: Fixed cells

A

Direct detection

Amplified or indirect detection

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)

14
Q

Amplifies detection

A

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

15
Q

Bursting

A

switching between on and off states

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

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
Q

fluorescent protein detection

A

1) use brighter fluor
2) sensitive microscope methods(TIRF)
3) Time inebriation can help, if applicable

26
Q

gfp imaging

A

allows detecting stochastic decision making thresholds

27
Q

optical trapping

A

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
Q

DNA curtains

A

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
Q

Helicos-single molecule methods

A

direct imaging of fluorescent reversible terminators in single elongating DNA molecules

30
Q

Pacific Bioscience-single molecule methods

A

Residence time of flurorescently labeled dNTPs at immobilized polymerase

31
Q

Oxford nanopore-single molecule methods

A

pass single molecules through pore and measure electrical signal