Methods used to study gene expression

1. Methods used to study gene expression Slot blots Northern blots In situ hybridization RNA protection assay Primer extension dd-RTPCR RT-PCR Quantatitative RT-PCR (real-time) Microarrays

2. Northern Blotting • Detection of specific RNA molecules • Isolate total RNA • Purify poly A+ RNA if necessary • Separate RNA fragments by Agarose Gel Electrophoresis • Visualise • Blot onto Membrane

3. Probe for specific fragments (RNA molecule) • Label probe • Hybridise to membrane • Carry out washing at desired stringency • Detect using suitable system

4. Ribosomal RNA tRNA

5. 5 7 10 14 20 Western and Northern analysis of alternative oxidase expression AOX3 Western AOX2 AOX3 Northern AOX2 Northern Cotyledon Age (days)

6. Probe - Gene of Interest If known fine but what if do not have sequence Use heterelogous Probe ? Clone Gene of interest - variety of means

7. Label Probe • Variety of methods • Nick Translation • Random primer labelling with Klenow • PCR labelling • Label with What ? • Nucelotide that has a tag • Chromogenic • Radioactive • Luminescence • Fluorescence

8. Detection • All comes down to sensitivity • How much of the molecule will be present and is you labelling and detection system sensitive enough to detect • Sensitivity of Northern blotting is an issue • Amount of RNA required is large • How comparative are different blots - can different blots on same gene be compared and can gene be compared • These issues have led to the development of other approaches to measure gene expression

9. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Reverse Transcriptase Scheme

10. RT-PCR and biological relevance • RT-PCR (micro arrays, northern blots) measure steady state mRNA levels • RT-PCR (micro arrays, northern blots) do NOT measure transcription, mRNA stability or gene expression levels

11. DNA amplification: Polymerase Chain Reaction (PCR) heat to separate, add primers DNA synthesis from primers Cycle 2 etc, etc, etc, etc, DNA synthesis from primers Cycle 1 + DNA polymerase dATP, dCTP, dGTP, dTTP heat to separate, add primers fragment of chromosomal DNA after 15 cycles, 32,768 copies; after 30 cycles, 1,073,741,824 copies

12. Theoretical Real Life Log Target DNA Reality Check • Exponential increase is limited • Linear increase follows exponential • Eventually plateaus Cycle #

13. The starting material for gene expression studies is RNA. For real time RT-PCR EVEN quality of RNA required. The RNA can either be: • Total RNA • mRNA (poly A+)

14. What is Total RNA? -All available RNA in the cell. rRNA: Building of ribosomes: machinery for synthesizing proteins by translating mRNA. Main constituent of total RNA. 4 kinds, in eukaryotes, these are 18S rRNA, 28S, 5.8S, and 5S rRNA mRNA: Translated into a polypeptide. mRNA can be purified using oligo d-T primers attached to a resin (polyA purified RNA) tRNA: RNA molecules that carry amino acids to the growing polypeptide. snRNA: The primary transcripts for mRNA, rRNA, and tRNA produces large precursor molecules, must be processed within the nucleus to produce the functional molecules for export to the cytosol. Some of these processing steps are mediated by snRNAs. snoRNA: RNAs that help process ribosomal RNA (rRNA) molecules. miRNA: These are tiny (~22 nts) RNA molecules that appear to regulate the expression of mRNA molecules.

15. The RNA is transcribed to cDNA using reverse transcriptase and oligo dT primers, random hexamers or gene specific primers. This cDNA is then used in a real-time PCR reaction to determine the initial amount of RNA put in the RT (reverse transcriptase!) reaction. • What is the large assumption in making the above calculations?

16. The assumption: • All of the RT reactions occur at the same efficiency for all samples. Any problems with this?

17. RT-PCR Reverse transcription mRNA AAAAAAAAAAA TTTTTTTTTTTT cDNA formation Oligo dT primer PCR amplification cDNA TTTTTTTTTTTT F TTTTTTTTTTTT R PCR product F R

18. Reverse transcriptases MMLV (Moloney Murine Reverse Transcriptase): Lower activity temp; 37 C Lower intrinsic Rnase H activity AMV (Avian Myoblastosis Virus): Higher activity temp 41 C Higher intrinsic Rnase H activity Tth (therus thermophilus): Both RT and DNA polymerase High activity temp, 68-74 C Significant less efficient than either above

19. Quantifying cDNA accurately is difficult - lets you know how efficient RT was carried out • Direct - Spike with 32P-labeled dNTP (dCTP) precipitate and calculate mass of cDNA synthezised • Indirect - Determine Ct level for a specific sample and gene of choice and use that as a standard for further synthesis steps = Housekeeping gene (Normalise)

20. Pure RNA samples, genomic DNA contamination and pseudo genes • Treat samples when possible using RNase free DNase • Design primers to bind to different exons or if possible over exon/exon junctions

21. 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ The PCR Reaction d.NTPs Primers Add Master Mix and Sample Thermal Stable DNA Polymerase Add to Reaction Tube Denaturation Annealing

22. 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq 5’ The PCR Reaction Extension Extension Continued Repeat

23. 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ THE PCR REACTION Cycle 2 4 Copies Cycle 3 8 Copies

24. PCR - Powerful Tool!! • PCR technology is an essential tool for Molecular Biology • PCR allows rapid and reproducible amplification of a specific sequence of DNA • PCR technology is responsible for accelerating Genetic Discoveries • HOWEVER, IT COULD DO MORE!

25. Theoretical Real Life Log Target DNA Reality Check • Exponential increase is limited • Linear increase follows exponential • Eventually plateaus Cycle #

26. PCR - Powerful Tool!! • PCR technology is an essential tool for Molecular Biology • PCR allows rapid and reproducible amplification of a specific sequence of DNA • PCR technology is responsible for accelerating Genetic Discoveries • HOWEVER, IT COULD DO MORE!

27. Relative Fluorescence End Point Measurements In Addition

28. 96 Replicates of identical reactions have very different individual efficiencies by the end of the reaction

29. Threshold Cycle, Ct, of the same 96 replicates shows nearly identical values

30. What is Real Time PCR? Real Time PCR incorporates the ability to directly measure and quantify the reaction while amplification is taking place.

31. CT What is Threshold Cycle (CT)?

32. Threshold Cycle, Ct, is a reliable indicator of initial copy number

33. What Detection Strategies are available?

34. Dyes • Intercalating Dyes are inexpensive compared to hybridization probes. • A dye based strategy allows one to take a “big picture” - that is - get a general confirmation of amplification. • Russ Higuchi demonstrated the key principle of Real Time PCR using Ethidium Bromide - • EtBr fluoresces 25 times more brightly when bound to dsDNA • SYBR Green, a more sensitive dye is an even more attractive approach • SYBR Green fluoresces 200 times more brightly when bound to dsDNA

35. Sybr Green binding It can intercalate into DNA - but this does not result in increase in Flouresence Binding to minor groove of ds DNA results in Increase in Fluorescence Can bind ssDNA - no increase in binding Fluorescence can be quenched by impurities in RNA sample Binding influenced by: Salt concentration (NaCl, MgCl2) Concentration of ds DNA

36. 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ Taq ID 5’ 3’ 5’ 3’ Dyes d.NTPs Primers Intercalation Dyes Add Master Mix and Sample Thermal Stable DNA Polymerase Reaction Tube l Denaturation Annealing

37. 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq l l l ID ID ID ID ID ID ID ID ID ID 5’ l l Dyes Extension Extension Continued Apply Excitation Wavelength Repeat

38. Hybridization Probes Today Hybridization Probe Strategies fall into three main categories: • Cleavage Based Assay - TaqManä Assays • Displaceable Probe Assays • Molecular Beacons • Dual oligo FRET probes • Probes incorporated directly into the primers • Amplifluor • Scorpions