Methods used to study gene expression 4/22/2017 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

Northern Blotting Detection of specific RNA molecules 4/22/2017 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

Probe for specific fragments (RNA molecule) 4/22/2017 Probe for specific fragments (RNA molecule) Label probe Hybridise to membrane Carry out washing at desired stringency Detect using suitable system

4/22/2017 Ribosomal RNA tRNA

Western and Northern analysis of alternative oxidase expression 4/22/2017 Western and Northern analysis of alternative oxidase expression AOX3 Western AOX2 AOX3 Northern AOX2 Northern 5 7 10 14 20 Cotyledon Age (days)

Probe - Gene of Interest 4/22/2017 Probe - Gene of Interest If known fine but what if do not have sequence Use heterelogous Probe ? Clone Gene of interest - variety of means

Random primer labelling with Klenow PCR labelling 4/22/2017 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

4/22/2017 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

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 4/22/2017 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Scheme

RT-PCR and biological relevance 4/22/2017 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

Polymerase Chain Reaction (PCR) DNA amplification: Polymerase Chain Reaction (PCR) 4/22/2017 DNA synthesis from primers Cycle 1 + DNA polymerase dATP, dCTP, dGTP, dTTP heat to separate, add primers fragment of chromosomal DNA etc, Cycle 2 heat to separate, add primers DNA synthesis from primers after 15 cycles, 32,768 copies; after 30 cycles, 1,073,741,824 copies

Reality Check Exponential increase is limited 4/22/2017 Reality Check Exponential increase is limited Linear increase follows exponential Eventually plateaus Theoretical Real Life Log Target DNA The Theory of the amplification kinetics is much different then what is actually observed. Picture explains the actual kinetics of the PCR process. Remember, PCR is an enzymatic process and is therefore affected by the same factors that effect ANY Enzymatic process. Cycle #

4/22/2017 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+)

What is Total RNA? -All available RNA in the cell. 4/22/2017 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.

4/22/2017 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?

4/22/2017 The assumption: All of the RT reactions occur at the same efficiency for all samples. Any problems with this?

RT-PCR Reverse transcription PCR amplification F R F R 4/22/2017 mRNA AAAAAAAAAAA TTTTTTTTTTTT cDNA formation Oligo dT primer PCR amplification cDNA TTTTTTTTTTTT F TTTTTTTTTTTT R PCR product F R

Reverse transcriptases 4/22/2017 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

4/22/2017 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)

genomic DNA contamination and pseudo genes 4/22/2017 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

The PCR Reaction Add Master Mix and Sample Add to Reaction Tube 4/22/2017 The PCR Reaction 5’ 3’ 5’ 3’ 5’ 3’ d.NTPs 5’ 3’ Primers Add Master Mix and Sample 5’ 3’ Thermal Stable DNA Polymerase 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Add to Reaction Tube Most researchers are well versed in the performance of PCR. However, I usually emphasize that the performance of Real-Time PCR requires an emphasis on each step. STEPS 1) Prepare the Master Mixture and add all components to the reaction tube. 2) Denature the Template to generate single-stranded DNA. Usually performed at 95 oC for 30 to 60 s. 3) Annealing temperature. The temperature is lowered in order to promote the binding of the primers to its complimentary target. Generally ranges between 50 and 65 oC. 5’ 3’ 5’ 3’ Denaturation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Annealing

The PCR Reaction Extension Extension Continued Repeat Taq Taq Taq Taq 4/22/2017 The PCR Reaction 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Extension Taq 5’ 3’ 5’ 5’ 5’ 3’ Taq Extension Continued Starts with a Picture of the Annealed Template. 4) Extension Step - Between 65 and 72 oC. In many cases, this step is linked to the annealing temperature. When done this way, it is considered a 2 step PCR. Lead into next slide It is repeated to generate an exponential growth. Taq 5’ 3’ 5’ 5’ Taq 3’ Repeat

THE PCR REACTION Cycle 2 4 Copies Cycle 3 8 Copies 4/22/2017 5’ 3’ Cycle 2 4 Copies 5’ 3’ Cycle 3 8 Copies This demonstrates the power of exponential growth. Start with 2 copies, then 4 copies, then 8 copies, etc.

4/22/2017 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! If PCR is so powerful, then why should we improve it? PCR is used everywhere. In almost every laboratory of today. To increase the productivity of today's laboratory and to increase discoveries/advances.

Reality Check Exponential increase is limited 4/22/2017 Reality Check Exponential increase is limited Linear increase follows exponential Eventually plateaus Theoretical Real Life Log Target DNA The Theory of the amplification kinetics is much different then what is actually observed. Picture explains the actual kinetics of the PCR process. Remember, PCR is an enzymatic process and is therefore affected by the same factors that effect ANY Enzymatic process. Cycle #

4/22/2017 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! If PCR is so powerful, then why should we improve it? PCR is used everywhere. In almost every laboratory of today. To increase the productivity of today's laboratory and to increase discoveries/advances.

End Point Measurements 4/22/2017 End Point Measurements Relative Fluorescence Explanation of the plateau effect and the differences with the end point. Specifically relate the area under the square. If these points are used as a determination of starting copy number, we would be making the wrong determination. Lead into the next slide. Even worse with end point measurements. In Addition

4/22/2017 96 Replicates of identical reactions have very different individual efficiencies by the end of the reaction Look at the green circle. 96 replicates of the same sample yield different results for the end-point measurements. Where then can we measure the concentrations effectively?

4/22/2017 Threshold Cycle, Ct, of the same 96 replicates shows nearly identical values Explain the area shown as being in the exponential phase. This is the same slide as earlier. The variability of the end point fluorescence is not observed at the exponential phase. During the exponential phase 96-well replicates data show that all have similar behavior in exponential phase, but differ significantly at end of run We set out to devise methods to capture relevant quantitative data using the conventional PCR methods available prior to Real Time PCR The Ct level will be discussed in 2 slides.

4/22/2017 What is Real Time PCR? Real Time PCR incorporates the ability to directly measure and quantify the reaction while amplification is taking place. Self explanatory

What is Threshold Cycle (CT)? 4/22/2017 What is Threshold Cycle (CT)? CT The Ct discussed. Need to emphasize the steps. 1) Establish a baseline - Baseline value of no significant activity in the experimental parameters 2) Set Threshold - fluorescent level above baseline which indicates a positive signal is present. 2) Set the threshold (either you can do or the instrument will do based on preset settings). The Ct is the Point at which your sample crosses the threshold. General concept The higher the Threshold is set, the higher the CT observed. Many times this level is adjusted to account for noise in the reaction (I wouldn’t bring this up - just be aware of it).

Threshold Cycle, Ct, is a reliable indicator of initial copy number 4/22/2017 Threshold Cycle, Ct, is a reliable indicator of initial copy number Plot of Ct versus copy number. Note the Dynamic Range of 101 to 1010.

What Detection Strategies are available? 4/22/2017 What Detection Strategies are available? Getting ready to discuss Reaction Chemistries.

Dyes 4/22/2017 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 Intercalating dyes bind to double stranded DNA.

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

Dyes Add Master Mix and Sample Reaction Tube Denaturation Annealing l 4/22/2017 Dyes 5’ 3’ 5’ 3’ d.NTPs Primers Intercalation Dyes Add Master Mix and Sample Thermal Stable DNA Polymerase Reaction Tube Same basic format as with standard PCR. However, the Intercalation Dye is also included. Very low background when not bound to dsDNA. 5’ 3’ Taq 5’ 3’ l Denaturation 5’ 3’ 5’ 3’ ID 5’ 3’ 5’ 3’ Annealing

Dyes Extension Extension Continued Apply Excitation Wavelength Repeat 4/22/2017 Dyes 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Extension Taq 5’ 3’ ID ID 5’ 5’ ID ID ID 5’ 3’ Taq Extension Continued Apply Excitation Wavelength l The intercalation dye begins adding at the Extension step. When the proper wavelength is applied, the fluorescence of bound dyes is very high over background. Sybr Green = 495 nm Taq 5’ 3’ ID 5’ 5’ Taq 3’ l Repeat

Hybridization Probes Today Hybridization Probe Strategies 4/22/2017 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 Types of Hybridization probes.

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Identify promoter motifs that distinguish gene clusters 4/22/2017 Identify promoter motifs that distinguish gene clusters

TaqManTM Add Master Mix and Sample Reaction Tube Denaturation 4/22/2017 TaqManTM 5’ 3’ 5’ 3’ d.NTPs Primers Add Master Mix and Sample Thermal Stable DNA Polymerase 5’ 3’ R Q Probe Reaction Tube Again, same as the basic PCR reaction. However, a TaqMan probe is added. Note that the reaction is quiet when intact. 5’ 3’ Taq 5’ 3’ Denaturation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ l 5’ 3’ R Q Annealing

TaqManTM Extension Step 1. Strand Displacement 2. Cleavage 4/22/2017 TaqManTM 5’ 3’ R Q 5’ 3’ 5’ 3’ Extension Step R Taq 3’ Q R 1. Strand Displacement 5’ 5’ 3’ R Taq 3’ Q 2. Cleavage 5’ 5’ 3’ Taq R Q The Extension and detection step is a 4 step process. 1) Strand Displacement - The probe and Taq Polymerase comes in contact. 2) Cleavage - The probe is cleaved through the 5’ to 3’ exonuclease activity of Taq DNA polymerase. 3) Polymerization Complete - Probe is entirely cleaved. Extension is complete. 4) Detection - Apply proper wavelength and detect signal. 3. Polymerization Complete 3’ 5’ 5’ 3’ 5’ 3’ Q Taq R l 4. Detection

Molecular Beacons Add Master Mix and Sample Reaction Tube Denaturation 4/22/2017 Molecular Beacons 5’ 3’ d.NTPs Thermal Stable DNA Polymerase Primers Add Master Mix and Sample R Q Molecular Beacon Reaction Tube Same basic format as standard PCR. Denaturation 5’ 3’ Taq R Q Annealing

Molecular Beacons Extension Step Detection 1. Strand Displacement 4/22/2017 Molecular Beacons l 5’ 3’ R Q Detection 5’ 3’ Extension Step 5’ 3’ R Q Taq 5’ 1. Strand Displacement 5’ 3’ 5’ 3’ Taq R Q Molecular Beacon 2. Polymerization Complete Probe Silent Detection is performed at the annealing step due to the opening of the probe. The binding and unfolding of the probe is because it is energetically more favorable to bind the target sequence than to remain in the folded conformation.

FRET Probes Extension Step Detection 1. Strand Displacement 4/22/2017 FRET Probes l 3’ D R 5’ 5’ 3’ Detection 1-5 bases Extension Step R 3’ D Taq 5’ 1. Strand Displacement System Silent R 5’ 5’ 3’ D R 2. Polymerization Complete System Silent This approach requires the binding of probes closely together (within 1-5 bases). Differs form other approaches in that the energy is transferred from the first fluorophore to the second. The fluorescence of the second fluorophore is then analyzed. 5’ 3’ Taq

Primer Based Heat Incorporation l l 4/22/2017 3’ 5’ R Q l R Q Heat Incorporation Essentially a molecular beacon attached to a primer. Follows the same basic principle as molecular beacons for measuring fluorescence. l

Primer Based Annealing/Extension 1 Extension 2 Detection l 4/22/2017 5’ 3’ R Q Annealing/Extension 1 3’ 5’ R Q Extension 2 5’ The primer gets incorporated during the first annealing/extension step and is still “silent”. The primer gets unfolded during the 2nd extension step and signal can now be achieved. Problems with primer dimers will result in an increased background signal. l Q R 3’ 5’ Detection