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
