Analysis of gene expression using RT-PCR Dr. Tamas Borsics ABE Workshop 2006 Leeward Community...
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Transcript of Analysis of gene expression using RT-PCR Dr. Tamas Borsics ABE Workshop 2006 Leeward Community...
Analysis of gene expression using RT-PCR
Dr. Tamas Borsics
ABE Workshop 2006 Leeward Community College
9:30 am Friday, June 16, 2006
What does the term “RT-PCR” stand for?
Involves two processes:
RT – Reverse Transcription
During this step we synthesize single stranded DNA from RNA template
PCR – Polymerase chain reaction
Using gene-specific primers we amplify a certain part of our gene of interest to get enough amount for further analysis
DNA: Double stranded – very stable, durable
• Used in cells to store information (chromosomes)
• Material of inheritance
• Used in forensic science
• Used by archeologists (~ 50.000 years)
DNA and RNA – What a difference!
RNA: single stranded – unstable, strong secondary structures
• Used in cells to temporary express information
• Cell’s RNA profile changes from minute-to-minute
• Degrades very easily – even during the isolation process
DNA and RNA – What a difference!
The DNA replicates its information in a process that involves many enzymes
The DNA codes for the production of messenger RNA (mRNA)
In eucaryotic cells, the mRNA is processed (essentially by splicing) and migrates from the nucleus to the cytoplasm
Messenger RNA carries coded information to ribosomes. The ribosomes "read" this information and use it for protein synthesis
The Central dogma of Molecular BiologyU
NID
IRE
CT
ION
AL
What makes the difference?
Human: 30 000Roundworm: 19 000
Fruit fly: 13 500Arabidopsis: 25 000
Yeast: 6 300E. coli: 3 200
# of genes
DNA content is the same in each cell type
RNA profile is different in each cell type
Each cell uses (transcribes) only a small fraction (10-15%) of all the genes in the genome into RNA
How transcription works?
Transcription begun and mRNA elongation is underway
Transcriptase enzyme binds to the double stranded DNA, separates strands and the DNA 'eye' opens.
Elongation continues till the end of the transcription unit
RNA viruses (Retroviruses)
Viruses
The discovery of reverse transcriptase
Double stranded DNA viruses
Single stranded DNA viruses
• Do not contain DNA
• first recognized to cause certain cancers in animals
• Tumor cells have genetic-like stability
• RNA is not suitable for stable transfection
HOW RNA tumor viruses cause cancer???
The discovery of reverse transcriptase
Already known: certain bacterial viruses could integrate their DNA into their hosts' chromosomes and persist as stable genetic elements
Howard Temin proposed that RNA tumor viruses can cause permanent alterations to cells by integrating into host chromosomes.
RNAs first has to be converted into DNAs, which could then become integrated.
If all the above is true there MUST be a mechanism to convert RNA into DNA !
Is the Central dogma a “holy cow”?
?is there a way back???
Two separate research teams, one led by Temin and the other by
David Baltimore, simultaneously discovered the elusive RNA-copying
DNA polymerase in purified virions – after many years of painstaking
laboratory work.
The discovery of reverse transcriptase – the breaktrough
1970:
1975:
Temin and Baltimore shared the Nobel
Prize in physiology or medicine for their
discovery of reverse transcriptase.
Reverse transcriptase uses a single-stranded RNA template to create a double-stranded DNA.
What does reverse transcriptase do?
copy
c c c
How can it be used in research?
transcription
transcription
transcription
AAAAA
AAAAA
AAAAA
mRNA
mRNA
mRNA
• Excellent tool to study genes that are actually expressed in a certain type of cell/tissue/organ.
• Monitor gene expression changes due to environmental effects
Reverse transcription
cDNA analysis
Let’s start!
total RNA
tRNArRNAmRNA
~ 1%
• Most of the RNA is unimportant for us (tRNA, rRNA)
• mRNA population consists of about 3-5000 different kind
• Strong secondary structure – enzyme cannot work
AAAAAOnly mRNA has a poly-Adenin tail at the 3’ end
RNA isolation
RT reaction – step by step
AAAAA
AAAAA
AAAAA
65 ºC + ice
37 ºC
TTTTT
TTTTT
TTTTT
TTTTT
Reverse transcriptase
dATPdCTPdGTPdTTP
RNase inhibitor
mRNA
mRNA
1. denature
AAAAATTTTTmRNA
2. anneal + elongate
TTTTTcDNA AAAAA
37 ºC
RT
cDNART RT
ready
What shall we do with our cDNA now?
Problems:
• Vast variety of cDNA molecules (at least 3-5000 different kind)
• Only one copy DNA per mRNA strand exist
• Abundant (housekeeping) genes give 90% of the mRNA population
• They mask our gene of interest
Solution: we need a reaction that is
• able to amplify our low-abundance gene AND
• specific enough not to amplify other cDNAs
+++ Plus we need an enzyme that is able to copy DNA
The DNA replicates its information in a process called “replication”
DNA copying in a test tube
Can we use it on a cDNA template?
The enzyme who does the job:DNA-DNA polymerase
5` 3`
How DNA-DNA polymerase works
5`3` 5`
3`
5`Oligomer
- what if we denature and attach an oligo facing BACKWARDS…
1985: Kary B. Mullis
5`Pol
3`
5`3`
5`3`
5` oligomer
Pol
5` oligomer
We made one copy strand!
We made a second one!!
… and repeat it over and over again?
5` 3`
Pol
Polymerase Chain Reaction (PCR)
3 cycles – 2 4 cycles – 4 5 cycles – 8
..
10 cycles – 256..
20 cycles – 4 194 304..
30 cycles – 4 294 967 296
Cycle #4
Cycle #5
Kary B. Mullis: “what if we attach an oligo facing backwards?”
1993: Nobel Prize in Chemistry
1985:
Having good times - and inventing PCR
– an idea conceived while cruising the Pacific Coast Highway from San Francisco to Mendocino on a motorcycle.
How to amplify our gene of interest from the cDNA “soup”?
TTTTTcDNA AAAAA
TTTTTAAAAAcDNA TTTTT
AAAAAcDNA
PCR
Gel visualization
Gene #2
750 bp
500 bp
Gene #1
Gene-specific primers
RT–PCR at the bench
total RNA + oligodT
37 ºC – 1 hour
anneal + elongate
65ºC – 10 min
denature
Add:
Enzyme
dNTPs
RNasin RT ready
RT:
PCR:
DNA pol
dNTPs
primers
Buffer
MgCl2
95ºC3 min
denature amplify
95ºC – 30 sec55ºC – 30 sec72ºC – 1 min
72ºC10 min
finish
PCR ready
template
1-5 ul
Gel analysis
30 cycles
Applications of RT-PCR
• Cloning genes’ expressed forms (not genomic version)
• Monitor a gene’s expression level in any tissue
• Monitor expression changes following treatments
• Sophisticated RT-PCR: The real time PCR
• sequencing a whole mRNA profile
• EST (Expressed Sequence Tags) – database
• Microarray (DNA chip)
• Diagnose and easily differentiate between different cancer types
• Early detection of hidden illnesses
• etc…
Questions?
Resources, references
http://www.bookrags.com/sciences/genetics/reverse-transcriptase-gen-04.html
vcell.ndsu.nodak.edu/~christjo/vcell/animationSite/transcription/movie.htm
http://www.ambion.com/techlib/basics/rtpcr/index.html
….should you need more info just google around. RT-PCR is everywhere on the Web…
http://en.wikipedia.org/wiki/RT-PCR
Denaturation - separate parent strands in preparation new strand synthesis
Annealing - “stick” primers to the parent strands to prime DNA synthesis
Extension - addition of nucleotides, one at a time, to the growing end of the DNA strand (3’ end) using the parent strand as the template
PCR – A quick overview