Real-Time PCR

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David A. Palmer, Ph.D.Technical Support, Bio-Rad LaboratoriesAdjunct Professor, Contra Costa College

Real-Time PCR

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Objectives Today we’ll talk about Real-Time PCR:

1. What is real-time PCR used for?

2. How does real-time PCR work?

3. What chemicals and instruments are used to detect DNA?

4. What does real-time data look like?

5. How can we demonstrate real-time PCR in the classroom?

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Part 1: What is Real-Time PCR and what is it used for?

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What is Real-Time PCR?

PCR, or the Polymerase Chain Reaction, is a process for the amplification of specific fragments of DNA.

Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses.

As we will see, Real-Time PCR allows us to measure minute amounts of DNA sequences in a sample!

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What is Real-Time PCR? Conventional PCR

tells us “what”.

Real-Time PCR

tells us “how much”.

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What is Real-Time PCR used for?

Real-Time PCR has become a cornerstone of molecular biology. Just some of the uses include:

• Gene expression analysis–Cancer and Drug research

• Disease diagnosis and management–Viral quantification

• Food testing–Percent GMO food

• Animal and plant breeding–Gene copy number

• Forensics–Sample identification and quantification

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Real-Time PCR in Gene Expression Analysis

Example: BRCA1 Expression Profiling

BRCA1 is a gene involved in tumor suppression.

BRCA1 controls the expression of other genes.

In order to monitor level of expression of BRCA1, real-time PCR is used.

DNA

mRNA

Protein

BRCA1Real-T

ime P

CR

Determine gene expression and publish scientific paper!

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Real-Time PCR in Disease Management

Example: HIV Treatment

Drug treatment for HIV infection often depends on monitoring the “viral load”.

Real-Time PCR allows for direct measurement of the amount of the virus RNA in the patient.

Viral RNAMeasure amount of virus, adjust prescriptions.

Real-Time PCR

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Real-Time PCR in Food Testing

Example: Determining percentage of GMO food content

Determination of percent GMO food content important for import / export regulations.

Labs use Real-Time PCR to measure amount of transgenic versus wild-type DNA.

wt DNAGMO DNA

Real-Time PCR

International shipments depend on results!

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Real-Time PCR in Forensics

Example: Real-Time PCR in Forensic Analysis!

Stain Identification:New Real-Time methods can be directly used to identify the composition of unknown stains, with much better accuracy than traditional “color-change” tests.

DNA Quantification:Since standard forensic STR Genotyping requires defined amounts of DNA, Real-Time PCR can be used to accurately quantify the amount of DNA in an unknown sample!

What is it ??

Enough DNA to ID ??

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Part 2: How does Real-Time PCR work?

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How does real-time PCR work?

To best understand what real-time PCR is, let’s review how regular PCR works...

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The Polymerase Chain Reaction

How does PCR work??

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Thermal Stable DNA Polymerase

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Denaturation

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Annealing

Add to Reaction Tube

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How does PCR work??

Extension

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How does PCR work??

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Cycle 2

4 Copies

Cycle 3

8 Copies

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How does Real-Time PCR work?

…So that’s how traditional PCR is usually presented.

In order to understand real-time PCR, let’s use a “thought experiment”, and save all of the calculations and formulas until later…

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Imagining Real-Time PCR

To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25…

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What’s in our tube, at cycle number 25?

A soup of nucleotides, primers, template, amplicons (the amplified DNA product), enzyme, etc.

1,000,000 copies of the amplicon right now.

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How did we get here?

What was it like last cycle, 24?

Almost exactly the same, except there were only 500,000 copies of the amplicon.

And the cycle before that, 23?

Almost the same, but only 250,000 copies of the amplicon.

And what about cycle 22?

Not a whole lot different. 125,000 copies of the amplicon.

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If we were to graph the amount of DNA in our tube, from the start until right now, at cycle 25, the graph would look like this:

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So, right now we’re at cycle 25 in a soup with 1,000,000 copies of the target.

What’s it going to be like after the next cycle, in cycle 26?

?

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So where are we going?

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Probably there will be 2,000,000 amplicons.

And cycle 27?

Maybe 4,000,000 amplicons.

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Probably there will be 2,000,000 amplicons.

And cycle 27?

Maybe 4,000,000 amplicons.

And at cycle 200?

In theory, there would be 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 amplicons…

Or 10^35 tons of DNA…

To put this in perspective, that would be equivalent to the weight of ten billion planets the size of Earth!!!!

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A clump of DNA the size of ten billion planets won’t quite fit in our PCR tube anymore!!!

Realistically, at the chain reaction progresses, it gets exponentially harder to find primers, and nucleotides. And the polymerase is wearing out.

So exponential growth does not go on forever!

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If we plot the amount of DNA in our tube going forward from cycle 25, we see that it actually looks like this:

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MeasuringQuantities

How can all this be used to measure DNA quantities??

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MeasuringQuantities

Let’s imagine that you start with four times as much DNA as I do.

Picture our two tubes at cycle 25 and work backwards a few cycles.

Cycle Me You

25 1,000,000 4,000,000

24 500,000 2,000,000

23 250,000 1,000,000

Cycle 25

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MeasuringQuantities

So, if YOU started with FOUR times as much DNA template as I did…

…Then you’d reach 1,000,000 copies exactly TWO cycles earlier than I would!

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MeasuringQuantities

What if YOU started with EIGHT times LESS DNA template than I did?

Cycle Me You

25 1,000,000 125,000

26 2,000,000 250,000

27 4,000,000 500,000

28 8,000,000 1,000,000

Cycle 25

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MeasuringQuantities

What if YOU started with EIGHT times LESS DNA template than I did?

You’d only have 125,000 copies right now at cycle 25…

And you’d reach 1,000,000 copies exactly THREE cycles later than I would!

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MeasuringQuantities

We can easily see that the left-right shift in the curves is related to the starting quantity of DNA!

Cq (“Cycle Quantity) values identify the curve positions, based on where they cross a threshold.

DNA Quantity and Cq value are related as:

Quantity ~ 2Cq

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MeasuringQuantities

We can plot the Cq value versus the Log Quantity on a graph…

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Log Quantity

Ct

4 unitsCq=23

1 unitCq=25

0.125 unitCq=28

… and calculate the quantity of any ‘unknown’ right off of the line!!

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Real-Time PCR

Sensitivity

How sensitive is Real-Time PCR?

Ultimately, even a single copy can be measured! In reality, typically about 100 copies is around the minimum amount.

One hundred copies of a 200-bp gene is:

• twenty attograms (2 x 10-17 g) of DNA!

• this is just 2/100ths of a microliter of blood!

Copy Number vs. Ct - Standard Curve

y = -3.3192x + 39.772

R2 = 0.9967

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Log of copy number (10n)

Ct

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Part 3: How do we detect and measure DNA?

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How do We Measure DNA in a PCR Reaction?

We use reagents that fluoresce in the presence of amplified DNA!

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Measuring DNA: Ethidium Bromide

Ethidium Bromide+ = common and well known

- = toxic, not very bright

http://www.web.virginia.edu/Heidi/chapter12/chp12.htm

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Measuring DNA: SYBR Green I

SYBR Green I+ = Bright fluorescence!

+ = Low toxicity!

Ames test results from Molecular ProbesSinger et al., Mutat. Res. 1999, 439: 37- 47

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Fluorescent Dyes in PCR

Intercalating Dyes

PCR makes more double-stranded DNA

SYBR Green dyebinds to dsDNA

When illuminated with light at 490nm, the SYBR+DNA complex fluoresces at 520nm.

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SYBR Green in Action!

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Fluorescent Dyes in PCR

Other Options

Even more ways to detect PCR products:

•Other Intercalating Dyes- Eva Green

•Probes- TaqMan Probes

•Primer/Probe Combinations- Scorpions- LUX Primers

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What Type of Instruments are used with Real-Time PCR?

What about the Instruments?

Real-time PCR systems consist of THREE main components:

1. Thermal Cycler (PCR machine), linked to a…

2. Optical Module (to detect fluorescence in the tubes during the run), linked to a…

3. Computer (to translate the fluorescence data into meaningful results).

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A good example is the MiniOpticon real-time instrument.

Optical ModuleThermal Cycler Base

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The CFX module scans the PCR plate with LEDs and records fluorescence in each well at each PCR cycle.

One more example is the Bio-Rad CFX-Touch real-time PCR instrument.

Optical ModuleThermal Cycler Base

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What Type of Software is used with Real-Time PCR?

The computer, running real-time software, converts the fluorescent signals in each well to meaningful data.

Workflow:

1. Set up PCR protocol.

2. Set up plate layout.

3. Collect data.

4. Analyze data.

1 2 3,4

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Part 4: What does actual real-time data look like, and what are melt curves?

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Real-Time PCR

Actual Data

• This is some actual data from a recent real-time PCR run.

• Data like this can easily be generated by preparing a dilution series of DNA.

c366939

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Real-Time PCR

Final Product

• The final product of real-time PCR is a table of Ct values, from which amounts of DNA can be determined.

Well Fluor ContentCycle

Quantity ( Cq )

A03 SYBR Std-1 8.90

A04 SYBR Std-2 12.20






B03 SYBR Std-1 8.85

B04 SYBR Std-2 12.12





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Real-Time PCR

Melt Curves

• The fluorescence data collected during PCR tells us “how much” …

…. but there is another type of analysis we can do that tells us “what”!

c366939

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Melt Curves

Basics

Melt curves can tell us what products are in a reaction.

• The principle of melt curves is that as DNA melts (becomes single stranded), DNA-binding dyes will no longer bind and fluoresce.

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Melt Curves

Basics

RFU vs Temp

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• Melt curves can tell us what products are in a reaction.

• PCR products that are shorter or lower G+C will melt at lower temperatures.

• Different PCR products will therefore have different shaped curves.

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Melt Curves

Typical Data

• For convenience, we typically view the derivative (slope) of the actual melt curve data.

• The resulting graph looks like a chromatogram, with peaks that represent different PCR products.

dRFU/dT

Slope vs. Temp

Teaching Tip:Use Melt Curves to bring up a good discussion of why different DNA sequences will “melt” at different temperatures! Talk about base-pairing, secondary structure, energy levels, etc!

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Melt Curves

High-ResolutionAnalysis

• The new field of Precision Melt Analysis even allows differentiation between PCR products based on a single-base pair mismatch!

• PMA/HRM is now used in mutation screening, detection of biological diversity, and genetic analysis.

PCR melt data from different organisms is first collected….

Then normalized….

Then the organisms are compared against each other.

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Part 5: How can we demonstrate real-time PCR in the classroom?

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CSI: MATERIALS REQUIRED

• To run the CSI Kit in real-time, only a few additional items are needed…

• iQ SYBR Green Supermix and appropriate tubes/caps

• A real-time PCR instrument

Crime Scene Investigator Kitin Real-Time

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CSI: INSTRUCTIONS

• An Application Note and a Starter Kit are available (166-2660EDU):

• The PCR reactions in the CSI kit can be run in real-time as an add-on to the regular kit.

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CSI: FINAL RESULTS

• The Crime Scene Real-Time Extension is great for first-time users!!

• Teach PCR, Real-Time, Melt Curves, etc!

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GMO: MATERIALS REQUIRED

• To run the GMO Kit in real-time, only two additional items are needed…

• iQ SYBR Green Supermix and appropriate tubes/caps

• A real-time PCR instrument

GMO Investigator Kitin Real-Time

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GMO: INSTRUCTIONS

• An Application Note and Starter Kit (166-2560EDU) are available:

• Basically, run GMO kit with real-time reagents on a real-time instrument.

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GMO: FINAL PRODUCT

• Ultimately, you can even calculate the percentage GMO content in actual food samples!

• The GMO Real-Time extension is perfect for more advanced users!

Food Plant Cq

GMO Cq

DeltaCq

Plant

DeltaCq

GMO

GMOPlant ddCq

Ratio2ddCt

Ratio%

100% GMO 23 27 0 0 0 1 100

Non-GMO 24 39 -1 -12 -11 0.0004 0.04

Product A 22 30 1 -3 -4 .0625 6.3

Product B 25 29 -2 -2 0 1 100

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Getting started with teaching Real-Time PCR in the classroom is easy !

1. Use the Crime Scene Real-Time starter kit.2. Use the GMO Investigator Real-Time kit.3. Use your own PCR primers and templates

with SYBR Green Supermix.

It’s nothing more complicated than using a slightly different polymerase supermix, correct plastics, and running on a real-time instrument !

Real-Time in the ClassroomGetting Started

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Why Real-Time in the Classroom is Cool!

Real-Time PCR in the classroom is “cool” for a number of reasons!

• Instant Results! You can see amplification within minutes of starting the PCR run. No waiting until next lab period and waiting for gels to run.

• What-Will-Happen-Next Factor! Because samples amplify at different times, many students will want to wait for “their” sample to amplify so they can see what happens!

• Demystifying the Black Box! Now students can see what happens inside the PCR machine!

• Finally a way to connect Computers and Biology! Many “computery” students get really excited to see the computer control of the instrument and data collection / analysis!

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Conclusions We’ve covered the following topics today:

1. What is real-time PCR used for?

2. How does real-time PCR work?

3. What chemicals and instruments are used to detect DNA?

4. What does real-time data look like?

5. How can we demonstrate real-time PCR in the classroom?

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Resources and References

• David [email protected]

• Bio-Rad Technical Support1(800)4BIORADconsult.bio-rad.com

• Bio-Rad Explorer website: www.explorer.bio-rad.com

• Bio-Rad Explorer email: [email protected]

• Crime Scene Investigator PCR Basics Kit Real-Time PCR Application NoteBulletin 166-2505

• GMO Investigator Kit Real-Time PCR Application NoteBulletin 166-2605

• Real-Time PCR Applications GuideBulletin 5279

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Real-Time PCRPractical Exercise!

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• Today we’ll use the DNA in the Crime Scene Kit to make some dilutions for our real-time experiment!

• Each workgroup will have DNA from the Crime Scene kit that has been diluted 1:10, 1:100, 1:1000, 1:10000, or undiluted. This is your “Unknown” DNA.

• Each workgroup will prepare four real-time PCR reactions:– Unknown DNA (replicate 1)

– Unknown DNA (replicate 2)

– Unknown DNA diluted 1:100 (replicate 1)

– Unknown DNA diluted 1:100 (replicate 2)

• If all goes well, you’ll be able to tell from the Cq values:– Which unknown DNA you started with,

– How accurate your pipetting is,

– Whether your mini-dilution series demonstrates high-efficiency PCR.

Today’s Experiment:An Overview

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• Step 1: DNA Dilutions– Dilute your “Unknown” DNA 1:100. – Mix 1 ul of your DNA (screw-cap tube labelled 1-5)

into 99 ul of water (screw-cap tube labelled “W”).• Step 2: Prepare your PCR Tubes

– Add 20 ul of the spiked SYBR Green Supermix (from the screw-cap) tube labelled SMX to four PCR tubes.

• Step 3: Add DNA to your PCR Tubes– Add 20 ul of your DNA samples to each PCR tube:

• Unknown Replicate A• Unknown Replicate B• Unknown 1:100 Replicate A• Unknown 1:100 Replicate B

– Mix gently, avoiding bubbles!– Label appropriately.

• Step 4: Cap and Load the PCR Tubes – Place the optically-clear flat caps on the tubes.– Place your reactions in the real-time PCR machine.

Today’s Experiment:Step-By-Step

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• Our PCR protocol will look like this:• 1. 95C for 3 min (activates Taq)

• 2. 95C for 10 sec (denatures)

• 3. 52C for 30 sec (extend / anneal)

• 4. Plate read (captures fluorescence data)

• 5. Goto Step 2 for 39 more times

Today’s Experiment:PCR Protocol

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Real-Time PCR

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BONUS: How do we optimize Real-Time PCR and troubleshoot problems?

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• Optimization of real-time PCR reactions is important:

– Since real-time PCR calculations are based on a doubling of product every cycle, if the reaction isn’t optimized, this doubling will not occur.

Optimization

Why?

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• A well-optimized reaction will have evenly spaced standard curves with tight replicates:

• At 100% efficiency, 10-fold serial dilutions will be spaced 3.3 cycles apart from each other.

Optimization

Example

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• Optimization is normally done as follows:

1. Design multiple primer sets.

2. Empirically test each primer set with a standard curve.

3. Select best primer set, then run a temperature gradient experiment to determine best annealing temperature.

4. Standard curves are ideal for assessing optimization.

Optimization

Basics

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• Why is it important for teachers to be able to solve real-time PCR problems?

•Help students have better success with their projects!

•Preventing problems at the start can help avoid lost experiments and reagents!

•Being able to explain unusual results leads to great teaching opportunities!

Trouble-shooting Skills

Why?

Teaching Tip:Very often a student’s first real-time data isn’t perfect – this makes for a great chance to teach better pipetting skillls, experimental design, etc!

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Trouble-Shooting

• A successful real-time PCR experiment will have the following characteristics:

Replicates are tightly clustered

Baselines are relatively flat

Dilution series has expected spacing

Plateau height doesn’t matter

Melt curve has one peak per product.

Curves are all S-shaped

Curves are smooth

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Trouble-Shooting

Replicates

• If replicates aren’t tightly clustered, suspect:– Pipetting error– Poorly optimized PCR reactions– Sample evaporation– Unknowns outside of range of detection– Instrument calibration

Replicates are tightly clustered

Replicates are not tightly clustered

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Trouble-Shooting

Baselines

• If baselines aren’t flat, suspect:– Sample evaporation– Bubbles– Reagents not thoroughly mixed– Baseline “window” not properly set

Baselines are relatively flat

Baseline not flat

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Trouble-Shooting

Dilutions

Dilution series has expected spacing

1000100

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Dilution series tightly compressed

Dilution series stretched out

• If the dilution series comes out “compressed” or “stretched”, suspect:– Pipetting– Too much DNA (for your assay)– PCR inhibitors– Too little DNA (for your assay)– Poor PCR efficiency

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Trouble-Shooting

Curve Shape

• If curves are not S-shaped, suspect:– Curves are not actual PCR products!– Sample evaporation– Fluorescence drift in unamplified samples– Something seriously wrong with assay

Curves are all S-shaped

Curves are not S-shaped

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Trouble-Shooting

Curve Shape

• If curves are not smooth, suspect:– Poor pipetting (bubbles)– Sample evaporation– Poor assay (low fluorescence reagents)– System malfunction (line noise)

Curves are smooth

Curves are not smooth

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Trouble-Shooting

Melt Peaks

• If melt curves have more than one peak:– More than one product– Possible normal primer-dimers– Using too low an annealing temperature– Primers need to be redesigned

Melt curve has one peak per product.

Melt curves have multiple peaks.

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Trouble-Shooting

Common themes in troubleshooting:

Care in pipetting.

Care in choice of plastics and sealing the plates.

Care in experimental design.

Use of Positive and Negative Controls.

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Teaching Tip:Use Real-Time PCR to teach the importance of properly designed experiments !!

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Real-Time PCR

Real-Time PCR

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