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Analysis of gene expression 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 . PCR – Polymerase chain reaction.

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Analysis of gene expression using RT-PCR

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  1. Analysis of gene expression using RT-PCR Dr. Tamas Borsics ABE Workshop 2006 Leeward Community College 9:30 am Friday, June 16, 2006

  2. 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 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

  3. 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)

  4. 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

  5. UNIDIRECTIONAL The Central dogma of Molecular Biology 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

  6. # of genes Human: 30 000 Roundworm: 19 000 Fruit fly: 13 500 Arabidopsis: 25 000 Yeast: 6 300 E. coli: 3 200 What makes the difference? 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

  7. 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 How transcription works?

  8. The discovery of reverse transcriptase Double stranded DNA viruses 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???

  9. 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 !

  10. Is the Central dogma a “holy cow”? is there a way back??? ?

  11. 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.

  12. What does reverse transcriptase do? copy c c c Reverse transcriptase uses a single-stranded RNA template to create a double-stranded DNA.

  13. transcription transcription transcription How can it be used in research? AAAAA mRNA mRNA AAAAA AAAAA mRNA 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

  14. Let’s start! total RNA RNA isolation ~ 1% rRNA mRNA tRNA • 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

  15. RT cDNA RT reaction – step by step TTTTT TTTTT AAAAA RNase inhibitor TTTTT TTTTT dATP dCTP dGTP dTTP 65 ºC + ice 1. denature AAAAA mRNA Reverse transcriptase RT 2. anneal + elongate 37 ºC AAAAA mRNA TTTTT cDNA AAAAA 37 ºC AAAAA TTTTT mRNA RT ready

  16. 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

  17. 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?

  18. 5` 3` 3` 5` 5` 3` Oligomer 5` 5` 3` 5` We made one copy strand! 3` oligomer Pol Pol Pol 5` 3` 5` oligomer 5` We made a second one!! 3` 5` How DNA-DNA polymerase works 1985: Kary B. Mullis - what if we denature and attach an oligo facing BACKWARDS… … and repeat it over and over again?

  19. Polymerase Chain Reaction (PCR) Cycle #4 3 cycles – 2 4 cycles – 4 5 cycles – 8 . . 10 cycles – 256 . . 20 cycles – 4 194 304 . . 30 cycles – 4 294 967 296 Cycle #5

  20. 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

  21. TTTTT AAAAA cDNA TTTTT cDNA AAAAA cDNA TTTTT AAAAA Gene-specific primers Gene #2 Gene #1 750 bp 500 bp How to amplify our gene of interest from the cDNA “soup”? PCR Gel visualization

  22. Add: 65ºC – 10 min RNasin dNTPs denature Enzyme 1-5 ul 95ºC – 30 sec 55ºC – 30 sec 72ºC – 1 min template 95ºC 3 min Buffer MgCl2 dNTPs denature amplify DNA pol primers Gel analysis RT–PCR at the bench RT: 37 ºC – 1 hour RT ready total RNA + oligodT anneal + elongate PCR: 72ºC 10 min PCR ready 30 cycles finish

  23. 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…

  24. Questions?

  25. 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…

  26. 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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