Week 2 - Transcriptomics Flashcards
What is the transcriptome?
- mRNA
- All expressed RNA
What is the translatome?
all translated mRNA
- mRNA in polysomes
What is the proteome?
all expressed proteins
Differential gene expression
Methods of transcript detect/quantification
- Northern Blot
- RT PCR and qPCR
- Microarray
Northern Blot
- Pour denaturing agarose gel that will denature DNA
- RNA has secondary structure and gel changes the
mobility of the mRNA as it moves through
– straight mRNA = slow
– small hairpin == faster
– large hairpin === fastest
- larger mass = slower travel through denaturing gel
- Transfer to nitrocellulose –> binds to nucleic acids
- Hybridize with labelled DNA
- Audioradiogram showing where radioactivity is on the filter
____ RNAs travel slower than ____ RNAs
long, short
advantages of norther blot:
observing RNA directly and not cDNA copy
- determine size of the transcript directly
disadvantages of norther blot:
- not very density and needs a lot of RNA sample
- not qualitative
basically obsolete
RT PCR
cDNA copy of mRNA population is required because PCr requires DNA polymerase
- DNA copy made using RT which also need DNA primer
- also can use oligo dT primer which hybridizes with the polyA tail
advantages of RT PCR:
not all RNA in the transcriptome has polyA tail
process of RT PCR
- design primers that can anneal to sequences present in transcript –> can amplify section of DNA
- PCR product with the cDNA sample to mRNA for one condition
power of PCR
- product is amplified geometrically due to chain reaction, cycle 0: 1, cycle 1: 2, cycle 2: 4, cycle 3: 8, etc….
RT monitoring of product production
- monitor using fluorescence
cT - when threshold is passed - more cDNA for a specific transcript in the population = the lower the cT
- less cDNA for specific transcript in pop = higher cT
Microarray
detects differential gene expression
microarray process
- radioactively labelled cDNA to mRNA
- after hybridization and autoradiography (black = radioactive, clear/white = non-radioactive), can use colour labelled cDNA and detect fluorescence
what is a protein detection/quantification method
western blot analysis
Western blot analysis requirements:
- membranes with proteins absorbed
- antibody that will detect chosen protein
Western blot analysis process
proteins travel on polyacrylamide gel and separate based on size
- remove gel
- place nitro-cellulose membrane (proteins here are all negatively charged)
- incubate with antibody and with bind to chosen protein
- wash off unbound antibody
- can visualize using chemiluminescence
2 goals of RNA Seq
- Count the relative number of transcripts in the sample
- determine the structure of the transcripts in the sample
3 methods of RNA seq
all depend on how DNA is sequenced
1. Direct RNA seq (Most recent) –> least manipulations
2. Long read RNA seq (newer) –> more manipulations
3. Short read RNA seq. (most established) –> more manipulations
process of direct RNA seq
uses dna or directly seq RNA
- adapter will bind to motor protein and push other strand through nano pore
- polyt tail anneal to polyA tail
- RNA is threaded through nano pore membrane where change in current of molecules is measured as moved through
Nanopore sequencing
- DNA molecule added adaptors
- Motor molecule binds
- SS DNA thread through pore
- can detect modification of transcripts
- when RNA being threaded through, length of polyA tail can be determined
Advantages of direct RNA seq.
- direct RNA seq.
- long reads (1000 bases)
- only manipulation is addition of adaptors
- characterize modification and polyA tail length
disadvantages of direct RNA seq.
- lower throughput (10^6 reads)
- higher error rate
- no amplification possible (larger RNA sample)
long read RNA seq.
- DNA molecule has adaptors
- DNA and DNA polymerase in a cell –> smart cell and ZMWs
- DNA sequence read –> continuous long read (CLR) seq, > 50 kb, longest possible reads
long read RNA seq process
- make cDNA copy of RNA
- Adaptor ligation onto cDNA copies
- PCR amplification, can increase # of molecules being seq.
- Size detection
- long read seq.
not directly seq. RNA
- unambiguous to isoform such that each read counted as 1 transcript
advantages of long read RNA seq
- long reads: complete transcripts end to end
- less manipulation than short reads
- can amplify cDNA
disadvantages of long read RNA seq
-lower throughput (106 reads)
- higher error rate
- seq a DNa copy and not original transcript
Short Read RNA seq.
- uses flowcell, can simultaneously seq. millions to billions of mol.
- labelled nucleotides with fluorophore and blocked 3’ end –> in cycle 1, nuc. will be recognizes, at end of cycle, 3’block is removed and flurophore, repeat with next base
Short Read RNA seq. process
- DNA molecule has adaptors added
- Sticks to oligo on a glass slide
- amplified to male cluster of slide –> clonally amplified through bridge amplification
- denatured into 2, SS copies - first seq. read
- reorientation of the DNA mol.
- Second paired seq. read
- template now binds to second oligo
reads approx. 100 nuc.
- produces ambiguous exon (unable to differentiate x/y transcripts)
- also produces unambiguous exons (can differentiate x/y transcripts)
Advantages of Short Read RNA seq.
- paired end reads
- two sequence files from alternate ends of the same DNA fragment made, therefore its KNOWN the come from same fragment
- large # of reads (109-1010)
- lower error rate
- amplify sample
Disadvantages of Short Read RNA seq.
- higher number of manipulations
- larger amount of computation required due to short reads
- seq. a DNA copy and not original transcript
transcript number
- gene is being transcribed at a constant rate
- transcript is degraded and has a constant half-life
when transcription shut off:
- rate of degradation depends on number of transcripts present
- exponential decay –> first order kinetics
rate of transcription = rate of degradation
(week 2 - slide 95 and 96)
sc RNA seq goals
- determine the polyA+ transcriptome of individual cells
- useful in the study of development and human disease
sc RNA seq process
- cells from suspension
- micro-article and lysis buffer
- barcoded bead primer move through microfluid device and form droplet
- cell lysis
- RNA hybridization –> adds 3 G’s
- break droplets
- RT with template switching –> adds 3 C’s to end before and then extends
- STAMPs - single cell transcriptome attached to a microparticle –> can be amplified in PCR
- Sequencing and analysis
- each mRNA is mapped to original cell of origin and gene of origin
- each cell’s pool of mRNA can be analyzed
- cDNA alignment to genome and group results by cell - count unique UMIs for each gene in each cell, create digital expression matrix
**BIG DATA
t-distributed stochastic neighbour embedded (t-SNE)
data analysis and visualization tool that serapes and cluster multidimensional DNA
- gives each point a position in a 2 or 3D map
- 2D space pushes dissimilar data away from one another and clusters similar data together
