Transcript Analysis of genes using RT-PCR

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 and RNA – What a difference!
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
The Central dogma of Molecular Biology
UNIDIRECTIONAL
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
What makes the difference?
# of genes
Human: 30 000
Roundworm: 19 000
Fruit fly: 13 500
Arabidopsis: 25 000
Yeast: 6 300
E. coli: 3 200
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?
Transcriptase enzyme binds to the double stranded
DNA, separates strands and the DNA 'eye' opens.
Transcription begun and mRNA elongation is underway
Elongation continues till the end of the transcription unit
The discovery of reverse transcriptase
Viruses
Double stranded DNA viruses
Single stranded DNA viruses
RNA viruses (Retroviruses)
• 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???
The discovery of reverse transcriptase – the breaktrough
1970:
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.
1975:
Temin and Baltimore shared the Nobel
Prize in physiology or medicine for their
discovery of reverse transcriptase.
What does reverse transcriptase do?
copy
c
c
c
Reverse transcriptase uses a single-stranded RNA template to create a
double-stranded DNA.
How can it be used in research?
AAAAA
mRNA
mRNA
mRNA
AAAAA
AAAAA
Reverse transcription
cDNA analysis
• 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
Let’s start!
total RNA
RNA isolation
~ 1%
mRNA
tRNA
rRNA
• 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
Only mRNA has a poly-Adenin tail at the 3’ end
AAAAA
RT reaction – step by step
TTTTT
TTTTT
AAAAA
RNase
inhibitor
TTTTT
TTTTT
1. denature
dATP
dCTP
dGTP
dTTP
65 ºC + ice
AAAAA
mRNA
RT
2. anneal +
elongate
Reverse
transcriptase
37 ºC
AAAAA
mRNA
TTTTT
cDNA
AAAAA
37 ºC
AAAAA
mRNA
cDNA
RT
TTTTT
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
DNA copying in a test tube
The DNA replicates its
information in a process
called “replication”
The enzyme who does the job:
DNA-DNA polymerase
Can we use it on a cDNA
template?
How DNA-DNA polymerase works
5`
3`
3`
5`
5`
3`
Oligomer
5`
5`
3`
Pol
5`
We made one copy strand!
3`
1985: Kary B. Mullis
- what if we denature and attach an oligo facing BACKWARDS…
5` oligomer
3`
5`
5` oligomer
We made a second one!!
3`
… and repeat it over and over again?
Pol
Pol
5`
Polymerase Chain Reaction (PCR)
Cycle #4
3 cycles –
4 cycles –
5 cycles –
Cycle #5
2
4
8
.
.
10 cycles –
256
.
.
20 cycles –
4 194 304
.
.
30 cycles – 4 294 967 296
Having good times - and inventing PCR
1985: Kary B. Mullis: “what if we attach an oligo facing backwards?”
– an idea conceived while cruising the Pacific Coast Highway from San
Francisco to Mendocino on a motorcycle.
1993: Nobel Prize in Chemistry
How to amplify our gene of interest from the cDNA “soup”?
TTTTT
cDNA
AAAAA
cDNA
TTTTT
AAAAA
Gene-specific
primers
TTTTT
cDNA
AAAAA
PCR
Gene Gene
#2
#1
750 bp
500 bp
Gel
visualization
RT–PCR at the bench
RT:
65ºC – 10 min
total RNA +
oligodT
denature
Add:
37 ºC – 1 hour
RNasin
dNTPs
Enzyme
anneal +
elongate
RT
ready
PCR:
template
Buffer
95ºC
3 min
MgCl2
dNTPs
95ºC – 30 sec
55ºC – 30 sec
72ºC – 1 min
72ºC
10 min
30 cycles
denature
amplify
PCR
ready
finish
DNA pol
primers
Gel analysis
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
http://en.wikipedia.org/wiki/RT-PCR
….should you need more info just google around. RT-PCR is everywhere on the Web…
PCR – A quick overview
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