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Leeward Community College
Analysis of gene expression using RT-PCR Dr. Tamas Borsics ABE Workshop 2006 Leeward Community College 9:30 am Friday, June 16, 2006
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RT – Reverse Transcription
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
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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 (~ years)
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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
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The Central dogma of Molecular Biology
The DNA replicates its information in a process that involves many enzymes UNIDIRECTIONAL 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
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What makes the difference?
Human: Roundworm: Fruit fly: Arabidopsis: Yeast: 6 300 E. 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
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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
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HOW RNA tumor viruses cause cancer???
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???
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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 !
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Is the Central dogma a “holy cow”?
is there a way back??? ?
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1970: 1975: The discovery of reverse transcriptase – the breaktrough
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.
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What does reverse transcriptase do?
copy c c c Reverse transcriptase uses a single-stranded RNA template to create a double-stranded DNA.
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Reverse transcription
How can it be used in research? transcription AAAAA mRNA mRNA transcription AAAAA AAAAA mRNA transcription 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
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Only mRNA has a poly-Adenin tail at the 3’ end
Let’s start! total RNA RNA isolation ~ 1% mRNA rRNA tRNA Most of the RNA is unimportant for us (tRNA, rRNA) mRNA population consists of about different kind Strong secondary structure – enzyme cannot work Only mRNA has a poly-Adenin tail at the 3’ end AAAAA
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Reverse transcriptase
RT reaction – step by step TTTTT TTTTT AAAAA RNase inhibitor TTTTT TTTTT dATP dCTP dGTP dTTP 65 ºC + ice 1. denature AAAAA mRNA RT Reverse transcriptase 2. anneal + elongate 37 ºC AAAAA mRNA TTTTT cDNA AAAAA 37 ºC AAAAA mRNA cDNA RT TTTTT RT ready
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What shall we do with our cDNA now?
Problems: Vast variety of cDNA molecules (at least 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
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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?
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How DNA-DNA polymerase works
5` 3` 5` 3` 5` Oligomer We made one copy strand! 5` Pol 3` 5` 3` 1985: Kary B. Mullis - what if we denature and attach an oligo facing BACKWARDS… 5` oligomer 5` 3` We made a second one!! 5` oligomer Pol 5` 3` … and repeat it over and over again? Pol
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Polymerase Chain Reaction (PCR)
Cycle #4 3 cycles – 4 cycles – 5 cycles – . 10 cycles – 20 cycles – 30 cycles – Cycle #5
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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
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Gene-specific primers
How to amplify our gene of interest from the cDNA “soup”? TTTTT AAAAA cDNA TTTTT AAAAA cDNA Gene-specific primers TTTTT cDNA AAAAA PCR Gene #2 750 bp 500 bp Gene #1 Gel visualization
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RT: PCR: RT ready PCR ready RT–PCR at the bench 1-5 ul 30 cycles
Add: Enzyme dNTPs RNasin 65ºC – 10 min denature 37 ºC – 1 hour total RNA + oligodT RT ready anneal + elongate template 1-5 ul PCR: amplify 95ºC – 30 sec 55ºC – 30 sec 72ºC – 1 min 72ºC 10 min 95ºC 3 min denature DNA pol dNTPs primers Buffer MgCl2 PCR ready 30 cycles finish Gel analysis
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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…
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Questions?
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Resources, references vcell.ndsu.nodak.edu/~christjo/vcell/animationSite/transcription/movie.htm ….should you need more info just google around. RT-PCR is everywhere on the Web…
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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
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