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ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑ ΡΑΜΠΙΑΣ ΘΕΟΔΩΡΟΣ

ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑ ΡΑΜΠΙΑΣ ΘΕΟΔΩΡΟΣ. RT-PCR and quantitative RT-PCR for studying gene expression . OVERVIEW. tissue. extract RNA. copy into cDNA (reverse transciptase). do real-time PCR or RT-PCR. analyze results. PCR – Polymerase chain reaction.

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ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑ ΡΑΜΠΙΑΣ ΘΕΟΔΩΡΟΣ

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  1. ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑΡΑΜΠΙΑΣ ΘΕΟΔΩΡΟΣ

  2. RT-PCR and quantitative RT-PCR for studying gene expression

  3. OVERVIEW tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR or RT-PCR analyze results

  4. 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

  5. cDNA synthesis

  6. 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

  7. Sampling and Template Preparation • Important to be familiar with general principles of working with RNA: • Avoid RNAses • Always wear gloves when handling reagents or equipment that will be used in the RNA extraction and reverse transcription procedures • RNAse-free water can be commercially purchased or nanopure water can be treated with diethyl pyrocarbonate (DEPC) http://www.promega.com/~/media/files/resources/product%20guides/rna%20analysis%20notebook/workingwithrna.ashx?la=en

  8. IMPORTANCE OF RNA QUALITY • Should be free of protein (absorbance 260nm/280nm) • Should be undegraded (28S/18S ~2:1) • Should be free of DNA (DNAse treat) • Should be free of PCR inhibitors • Purification methods • Clean-up methods

  9. 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

  10. 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…

  11. Commonly Used PCR Assays • Conventional PCR utilizes two primers and products are detected by gel electrophoresis “cPCR” Agarose gel electrophoresis following PCR Log Target Cycle Number http://www.idtdna.com/pages/docs/educational-resources/gel-electrophoresis.pdf

  12. Real Time PCR

  13. Commonly Used PCR Assays • Real-time PCR detects a fluorescent signal that is increased each time a template is copied; the fluorescent signal is monitored each cycle or in ‘real-time’ ∆ Fluorescence Threshold CT CT Cycle Number CT = The cycle that a PCR reaction crosses the designated threshold Also called cycle quantification (CQ) or crossing point (CP) http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

  14. Commonly Used PCR Assays • Quantitative PCR relies on the principal that the quantity of target at the start of the reaction is proportional to amount of product produced during the exponential phase Greater starting target Less starting target ∆ Fluorescence CT < CT

  15. Quantitative PCR – in depth • Major assay types • Fluorogenic 5’ Nuclease Assay • Basis of TaqMan® chemistry • Uses two primers and an internal hydrolysis probe • Most commonly used for fish health diagnostics • SYBR ® green dye chemistry • Increased fluorescence when bound to dsDNA • Slightly lower specificity • Costs less • May not be as sensitive as the 5’ nuclease assays http://www.clinical-virology.org/pdfs/PCR_experience.pdf

  16. Quantitative PCR – in depth Step 1: Anneal and Polymerization Energy from fluorophore transferred to quencher Forward Primer R Q Probe Reverse Primer Taq Polymerase R T Step 2: Strand Displacement Q R Step 3: Cleavage Polymerization Complete Q Probe must hybridize specifically for cleavage A probe is cleaved each time a target is copied Fluorogenic 5’ Nuclease Assay

  17. Quantitative PCR – in depth http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_083618.pdf • Dual-labeled internal hydrolysis probes • 5’ reporter dye (typically Fam/Vic etc.) • 3’ quencher (typically non-fluorescent) • Can order from a range of oligo companies • Many companies have proprietary modifications for internal hydrolysis probes • Minor Grove Binding (MGB) – Applied Biosystems Inc. • The MGB linker raises the melting temperature of the internal hydrolysis probe and increases probe specificity

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