Week 2 - Transcriptomics

Question 1

What is the transcriptome?

Answer 1

• mRNA
• All expressed RNA

Question 2

What is the translatome?

Answer 2

all translated mRNA
- mRNA in polysomes

Question 3

What is the proteome?

Answer 3

all expressed proteins

Question 4

Differential gene expression

Question 5

Methods of transcript detect/quantification

Answer 5

1. Northern Blot
2. RT PCR and qPCR
3. Microarray

Question 6

Northern Blot

Answer 6

1. 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

2. Transfer to nitrocellulose –> binds to nucleic acids
3. Hybridize with labelled DNA
4. Audioradiogram showing where radioactivity is on the filter

Question 7

____ RNAs travel slower than ____ RNAs

Answer 7

long, short

Question 8

advantages of norther blot:

Answer 8

observing RNA directly and not cDNA copy
- determine size of the transcript directly

Question 9

disadvantages of norther blot:

Answer 9

• not very density and needs a lot of RNA sample
• not qualitative
  basically obsolete

Question 10

RT PCR

Answer 10

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

Question 11

advantages of RT PCR:

Answer 11

not all RNA in the transcriptome has polyA tail

Question 12

process of RT PCR

Answer 12

• 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

Question 13

power of PCR

Answer 13

• product is amplified geometrically due to chain reaction, cycle 0: 1, cycle 1: 2, cycle 2: 4, cycle 3: 8, etc….

Question 14

RT monitoring of product production

Answer 14

• 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

Question 15

Microarray

Answer 15

detects differential gene expression

Question 16

microarray process

Answer 16

1. radioactively labelled cDNA to mRNA
2. after hybridization and autoradiography (black = radioactive, clear/white = non-radioactive), can use colour labelled cDNA and detect fluorescence

Question 17

what is a protein detection/quantification method

Answer 17

western blot analysis

Question 18

Western blot analysis requirements:

Answer 18

• membranes with proteins absorbed
• antibody that will detect chosen protein

Question 19

Western blot analysis process

Answer 19

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

Question 20

2 goals of RNA Seq

Answer 20

1. Count the relative number of transcripts in the sample
2. determine the structure of the transcripts in the sample

Question 21

3 methods of RNA seq

Answer 21

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

Question 22

process of direct RNA seq

Answer 22

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

Question 23

Nanopore sequencing

Answer 23

1. DNA molecule added adaptors
2. Motor molecule binds
3. SS DNA thread through pore

• can detect modification of transcripts
• when RNA being threaded through, length of polyA tail can be determined

Question 24

Advantages of direct RNA seq.

Answer 24

• direct RNA seq.
• long reads (1000 bases)
• only manipulation is addition of adaptors
• characterize modification and polyA tail length

Question 25

disadvantages of direct RNA seq.

Answer 25

- lower throughput (10^6 reads) - higher error rate - no amplification possible (larger RNA sample)

Question 26

long read RNA seq.

Answer 26

1. DNA molecule has adaptors 2. DNA and DNA polymerase in a cell --> smart cell and ZMWs 3. DNA sequence read --> continuous long read (CLR) seq, > 50 kb, longest possible reads

Question 27

long read RNA seq process

Answer 27

1. make cDNA copy of RNA 2. Adaptor ligation onto cDNA copies 3. PCR amplification, can increase # of molecules being seq. 4. Size detection 5. long read seq. **not directly seq. RNA** - unambiguous to isoform such that each read counted as 1 transcript

Question 28

advantages of long read RNA seq

Answer 28

- long reads: complete transcripts end to end - less manipulation than short reads - can amplify cDNA

Question 29

disadvantages of long read RNA seq

Answer 29

-lower throughput (106 reads) - higher error rate - seq a DNa copy and not original transcript

Question 30

Short Read RNA seq.

Answer 30

- 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

Question 31

Short Read RNA seq. process

Answer 31

1. DNA molecule has adaptors added 2. Sticks to oligo on a glass slide 3. amplified to male cluster of slide --> clonally amplified through bridge amplification - denatured into 2, SS copies 4. first seq. read 5. reorientation of the DNA mol. 6. 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)

Question 32

Advantages of Short Read RNA seq.

Answer 32

- 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

Question 33

Disadvantages of Short Read RNA seq.

Answer 33

- higher number of manipulations - larger amount of computation required due to short reads - seq. a DNA copy and not original transcript

Question 34

transcript number

Answer 34

- 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)

Question 35

sc RNA seq goals

Answer 35

1. determine the polyA+ transcriptome of individual cells 2. useful in the study of development and human disease

Question 36

sc RNA seq process

Answer 36

1. cells from suspension 2. micro-article and lysis buffer 3. barcoded bead primer move through microfluid device and form droplet 4. cell lysis 5. RNA hybridization --> adds 3 G's 6. break droplets 7. RT with template switching --> adds 3 C's to end before and then extends 8. STAMPs - single cell transcriptome attached to a microparticle --> can be amplified in PCR 9. 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 10. count unique UMIs for each gene in each cell, create digital expression matrix **BIG DATA

Question 37

t-distributed stochastic neighbour embedded (t-SNE)

Answer 37

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