Quantitative PCR Session 4: Plate Preparation and Data Analysis

Presented by:

Robert O'BrienTraining Specialist – Forensic Biology

Quantitative PCRSession 4- Plate preparation and Data analysis

• Importance of pipetting technique with Quantifiler

• Preparing standards• Preparing reactions• Avoiding and detecting contamination during

qPCR set up• Starting a run• Analyzing data• IPC• Using quantifiler results

Importance of pipetting technique with Quantifiler

• The standards in quantifiler are the basis by which all quantity determinations are made using the SDS software.

• All quantities of samples are calculated based on the curve that is made from the standards.

• Because of this great care must be taken when preparing the standards.

• Most problems associated with quantifiler runs and the subsequent data are due to problems with standards which are usually because of mistakes in pipetting.

Importance of pipetting technique with Quantifiler

• Tips for improving accuracy and precision when pipetting– 1) Prewet the Pipette tip

• Draw in and expel sample at least 3 times before drawing in sample for delivery

• Purpose is to prevent evaporation that can occur in the tip. Prewetting the tip increases the humidity within the tip and therefore reduces the amount of sample evaporation

Importance of pipetting technique with Quantifiler

• 2) Work at room temperature

– Allow liquids and equipment to equilibrate to room temperature

– Volume of sample delivered by air displacement pipettes varies with air pressure, relative humidity and vapor pressure of the liquid, all of these are affected by temperature. Working at a single constant temperature minimizes this variation

Importance of pipetting technique with Quantifiler

• 3) Examine the tip before dispensing the sample

– Look at the outside of the tip to see if there is any liquid present.

– Liquid on the outside of the tip will increase the amount of liquid being dispensed. The outside of the tip can be wiped before delivery of tip contents but this must be done carefully because if the material being used to wipe the tip contacts the tip opening it can absorb the liquid inside of the tip

Importance of pipetting technique with Quantifiler

• 4) Use Standard Mode pipettingChoose standard mode pipetting rather than reverse mode pipetting

Standard mode pipetting – Push button to first stop of pipette then place pipette tip in liquid approximately 4mm below surface. Slowly release button to draw up liquid. To dispense slowly push button to first stop and then push all the way to second stop.Speed of drawing up and dispensing liquid should be the same

Importance of pipetting technique with Quantifiler

• Reverse mode pipetting – Push button to second stop to draw up liquid and only push to first stop to dispense. (Reverse mode is used for liquids with a high viscosity)

Importance of pipetting technique with Quantifiler

• 5) Pause consistently after drawing up liquid.

– Pause with the tip in the liquid for one or two seconds after drawing up the sample. The amount of liquid in the tip “bounces” slightly when the plunger stops. Slow even plunger release and a consistent brief pause after drawing up the liquid minimizes errors resulting from the “bouncing”

Importance of pipetting technique with Quantifiler

• 6) Pull the Pipette straight out

– After drawing up the sample pull the pipette straight out.

– Do not allow the tip to touch the sides of the container.

– With very small volumes < 50µl – surface tension effects cause the sample volumes to vary if the exit angle varies. Touching the tip against the sides results in loss of sample.

Importance of pipetting technique with Quantifiler

• 7) Minimize handling the pipette tip

• Avoid handling the tip and set the pipette down between sample deliveries. Body heat transferred to equipment during handling disrupts temperature equilibrium. Already stated was that temperature can affect volume of sample delivered.

• In forensic science work, the pipette tip should never be handled.

Importance of pipetting technique with Quantifiler

• 8) Immerse the tip to the proper depth.

• Immerse the tip 2 to 5mm below the meniscus and well clear of the container walls and bottom when drawing up the sample.

• Inserting the tip too far into the liquid causes excess droplets of liquid to cling to outside of the tip. Pressing or resting the tip against the container walls or bottom restricts entry of sample

Importance of pipetting technique with Quantifiler

• 9) Use the correct pipette tip.

– Securely attach a tip designed for use with the pipette. Mismatching a tip and pipette or using poor quality tips can result in an inadequate seal between the pipette and the tip. Quality tips are flexible and have thin walls providing an airtight seal and more dependable delivery of the sample.

Importance of pipetting technique with Quantifiler

• 10) Use constant plunger pressure and speed.

• Depress and release the plunger smoothly with consistent pressure and speed for each sample.

Preparing standards•There are 8 standards that are run in duplicate on every plate. The highest concentration is 50ng/µl and the lowest concentration is 0.023ng/µl.

•The first standard is made with the DNA standard provided by Applied Biosystems that comes with the Quantifiler kit.

To this standard is added TE buffer or a TE buffer glycogen mixture.

(glycogen is added to the TE so the standards will last longer once made)

Preparing standards• From this initial standard and TE buffer mixture subsequent

standards are made following a dilution series.

Preparing standards• The H standard in Human kits contains 14 to 16 copies

of a diploid single copy locus.

• The H standard in the Y kit contains 7 to 8 copies of a haploid single copy locus.

Preparing reactions

• Each well in the 96 well plate must have 23µl of PCR primer and reaction mix and 2µl of sample to give a total volume of 25µl.

• The primer/reaction mix is made up with 10.5 µl of quantifiler Human Primer (or Quantifiler Y primer depending on assay being conducted) and 12.5 µl of quantifiler reaction mix per well.

• When calculating how much of each to use make sure to include samples, standards and controls and a few extra for pipetting error.

Preparing reactions• First step is preparation of Standards

– Label 8 tubes for the standard series– Vortex quantifiler human standard– Pulse spin– Prepare serial dilutions using the quantities in the

table previously shown– Remember the standards are done in duplicate for

each assay.– During the preparation of standards great care must

be taken when doing the dilutions. Change pipette tip when going from one standard to the next during dilutions.

Preparing reactions• Before making master mix swirl the reaction mix by

hand. Do not vortex the reaction mix bottle.

• Vortex the primer mix and pulse spin.

• Make up the master mix, 10.5µl of primer mix and 12.5µl of reaction mix per well.

• Briefly vortex the master mix and pulse spin

Dispense 23µl of master mix into each well being used

Preparing reactions

• To the appropriate wells add 2µl of standards that were made and to the other wells the samples

• Seal the plate with an optical adhesive cover, sealing all edges and between well spaces with sealing tool

• Remove any air bubbles that are in sample wells by centrifuging the plate. This is necessary because bubbles in plate can show up as small amounts of DNA when there is no DNA present.

Avoiding and detecting contamination during qPCR set up

• Avoiding contamination during Quantitation

• Even though the 2µl of sample being used for quantitation will not be carried to STR amplification it is still important to avoid contamination of one sample with another.

• If two samples are mixed during set up of the plate then the quantities will be wrong and the incorrect amounts of sample will be taken on to the amplification process.

• There can be cross contamination from one sample to another, contamination from an outside source from the person setting up the plate or from amplified DNA

Avoiding and detecting contamination during qPCR set up

Avoiding contamination during Quantitation

Ways to prevent sample to sample contamination

1 Clearly label extraction tubes and only have one tube open at a time when loading the plate.

2 Arrange extraction tubes in the same order as they are going into the plate and move the extraction tubes after the sample is loaded to a different location to avoid the same sample being used twice.

3 Strip caps can be used to cover a row of wells on the plate after they are loaded. This prevents loading the same row of wells twice. It also prevents passing over loaded wells with a tip that contains DNA.

Avoiding and detecting contamination during qPCR set up

Avoiding contamination during Quantitation

Ways to prevent sample to sample contamination

4 When loading samples load them as close to the bottom of the well as possible and do not depress the pipette any further

after it is pulled out of well.

5 Once the plate is loaded and sealed with optical adhesive cover do not tilt or turn the plate in such a way that would cause the samples to get onto the cover.

Avoiding and detecting contamination during qPCR set up

Preventing contamination from an outside source

• Always wear PPE while loading plates. Especially face masks and gloves.

• Ensure the area where the plate is being set up in has been cleaned with bleach solution before plate set up.

• Have a separate room for plate set up and where the instrument is located. The instrument is amplifying DNA so it should be in a room dedicated for amplification. Set up should be in a room where there is not amplified DNA.

Avoiding and detecting contamination during qPCR set up

Detecting contamination

• An optional blank can be loaded into the plate during plate set up.

• This is different from the extraction blank because this will specifically detect if there was any contamination introduced during plate set up or if the plate was contaminated before being used.

• The most common type of contamination problem with setting up plates is well to well contamination because unlike tubes it is difficult to cap each well after it is loaded.

• It is important to keep an eye on your results. If a sample that you expected results gives you a negative and the sample next to it gave extremely high results then it is possible that a well was double loaded and the next well was skipped.

Avoiding and detecting contamination during qPCR set up

Detecting contamination

• Another indication of doubling of samples in a well is if a sample is run for both Total Human and Y and the Y gives a much higher result than the Total Human then it is possible that the Y well was double loaded.

• A lot of ways to detect contamination comes from thinking about results obtained from quantitation and determining if the results make sense. This will become easier with experience.

Starting a run• Instrument operation

• Turn on the instrument and allow it time to start up

• Open the AB Prism 7000 or 7500 SDS software,

set up the plate document for the sample run. Usually there will be a template document that can be opened so only sample names will need to be filled in.

Make sure the correct template is selected. Templates may vary based on the assays being performed (Total human only or Human and Y combination plate).

Starting a run• Once the sample names are filled in on the Plate

document, then check the thermal cycler conditions.– Ist step should read 95ºC for 10 minutes– 2nd step 40 cycles at 95ºC– 3rd step hold cycle for one minute at 60ºC

– Verify that the sample volume is set to 25µl and that the 9600 Emulation box is checked.

– Save the Plate document when you have finished

Starting a run• For 7000 place a compression pad on top of sealed

plate, gray side down directly over the reaction wells.

• For the 7500 no compression pad is needed.

• Place the plate in the instrument and close the door and start the run.

Analyzing the Data• Once the run is complete the plate must be

analyzed

• First verify all the analysis settings• Threshold set at 0.20000• Baseline start cycle 6• Baseline end cycle 15• If any of these are different they must be

changed before analysis. If after plate is analyzed it is noticed these values are not correct then they can be changed and the plate reanalyzed.

Analyzing the Data• To view analysis settings before analyzing plate under

Analysis go to Analysis settings the screen should be the same as this one.

Analyzing the Data• If the plate is already analyzed then check here for analysis settings

Analyzing the Data• To analyze plate go to Analysis and select analyze or click green arrow shown below

Analyzing the Data

• After analysis of plate is complete the following should be reviewed

– The standard curve– The amplification plot– The report

Analyzing the Data• The standard curve

• The standard curve gives the following information– The R² value of regression line which shows how well

the line fits with values from standards – The slope which gives an indication of efficiency– The Y- intercept

To select the standard curve under Results tab got to Standard curve verify slope for human is between

-2.9 to -3.3 and for Y -3.0 to -3.6

R² should be > 0.98

Analyzing the Data

Analyzing the DataThe values for the R² , and slope should be in the ranges shown for the data to be used.

If these values are not within range then there may have been a problem when setting up the standards that made the standard curve. This reinforces how careful one must be when making standards since if these values are not in range the plate may not be able to be used and the procedure may have to be repeated.

Analyzing the Data• Only with the standards for a Y plate can an adjustment

be made.• The STD 8 or H for the Y kit only has 7 to 8 copies per

2µl of the haploid target locus.

• Because of stochastic effects when using such a low amount of DNA the CT values for this concentration of DNA are more variable. This variability can throw off the line of best fit and this in turn will throw off R² value.

• So the STD 8 or H for the Y kit can be dropped by omitting the wells and reanalyzing.

Analyzing the Data• To omit well select the well then go to Well inspector and

click on omit well

Analyzing the Data• After well is omitted under the Plate tab the well should be crossed

out. Notice that the arrow is green indicating reanalysis is necessary.

Analyzing the Data• Troubleshooting Amplification Plot

• R² value < 0.98– Quantity values entered for quantitation standards in

well inspector are incorrect– Serial dilutions were not made properly– Standards were not loaded correctly– Reaction for one of the standards failed

Slope not in range

Assay not set up correctly

Software not set up correctly

Reagents may be expired or wrong ones used

Instrument may malfunction

Analyzing the Data• Y intercept

– There is no fixed value for the Y intercept– The Y intercept is the expected CT value for a sample

that has a quantity of 1ng/µl

– The Y intercept may shift from one kit lot number to the next. The Y intercept from all runs performed with a certain kit lot number should be within an acceptable range of the intercept value when the kit was quality checked. The range is lab dependant.

Analyzing the Data• After the plate is analyzed then the amplification plot is

checked to see what samples gave results and which were negative for DNA.

• The amplification plot is a plot of ΔRn vs Cycle number.

• This screen shows the corresponding wells for the samples. Each individual sample can be checked to see if the sample crossed the Threshold or not. Samples not crossing the threshold are negative no DNA present.

• Also the IPC can be checked for all of the samples.

• All IPCs should cross unless inhibition occurred.

Analyzing the Data

Analyzing the Data• The individual wells can be selected one at a time

• Also putting the cursor on a plot will show which sample it corresponds too.

• Under Detector there is a drop down menu to select.

• ALL, IPC, Quantifiler Human, or Total Y

Analyzing the Data

Analyzing the Data• To see either the Quantifiler Human or IPC by itself then go to

Detector drop down box and select what plot you would like to view

Analyzing the DataThe report

The report will show the quantities calculated for the samples.

The IPC well has no value under quantity because no quantity calculations are made for IPC,

If more than one sample has the same name then they are grouped together by the software and the Mean value and Standard deviation for the samples are calculated.

During practice plates duplicating samples is a good way to test precision of pipetting technique.

Analyzing the Data

IPC• IPC – Internal PCR control

• IPC is included as part of each reaction well. It undergoes the same PCR reaction as the sample but because its target sequence does not exist in nature it does not compete with the primers and probes that target human DNA.

• However because IPC is undergoing PCR along with the sample anything that negatively affects PCR of sample will also affect PCR of IPC.

IPC• Since the IPC is affected by problems with PCR that

affect the sample it can be used as a quality control check.

• IPC can detect the following– Presence of inhibitors– Problems with Assay set up– Chemistry or Instrument failure

IPC• If the IPC crosses the threshold and the sample does not then the

sample is considered a true negative.

IPC• The CT range for the IPC VIC dye should be between 20 to 30,

IPC• If both the Human specific target and the IPC fail to

amplify it is not possible to determine if the negative is due to no DNA present to amplify or if it is due to something affecting the PCR reaction like inhibition.

• If inhibition is suspected then the sample can be cleaned.– Cleaning can be done by concentrating the sample.

Some inhibitors will pass through the filters during the washes.

– If the sample was already concentrated then it can be diluted. This action can reduce the concentration of the inhibitor and in turn reduce its effect on the PCR reaction.

IPC• If neither of these work then the sample can be

re-extracted using a cleaner extraction method. Some extraction techniques like organic extraction produces a cleaner end product. Inhibitors are usually removed during organic extraction.

• If this is not possible then a new sample may be taken from the evidence that may be free of dyes or dirt or other materials that could have caused the inhibition.

IPC

• If the IPC does not cross the threshold and the sample has a low CT value and a high ΔRn that means there is a lot of target DNA present. In this case the DNA may have out competed the IPC for certain components necessary for amplification.

IPC• Earlier it was said that the IPC and target DNA have

different primers so they did not compete with each other for those. However there can be competition for polymerase and other components like Mg+ needed for PCR.

• If the sample has a high CT value and a low ΔRn and the IPC did not amplify it could mean there was partial inhibition. Depending on how low or high the quantity is the sample may still amplify and give a result during STR amplification or the sample may have to be cleaned up before continuing with the DNA analysis process.

Using Quantifiler results

• Should a sample that gives a negative result be taken on for further DNA analysis?

• Some laboratories choose not too carry the sample any further in an attempt to conserve money and time. During quantifiler the sample is put through 40 cycles of PCR. The PCR that occurs in the next stage of DNA analysis only puts the sample through 28 cycles. Theoretically if a sample does not amplify in 40 cycles it will not amplify in 28 cycles.

• As a result a true negative quantifiler result will not be expected to generate a profile. The practice of dropping a sample after quantitation is laboratory dependant.

Using Quantifiler results• If after quantitation there is DNA in your sample then the

extract must be taken on to STR amplification.– The final value from quantitation will be in the units– ng/µl– This simply means that for every 1 µl of extract there

is a certain amount of DNA.– STR amplification kits require a certain amount of

input DNA to give optimum results. Meaning a full profile with good peak heights.

– Usually targeting an input of 1ng of DNA for STR amplification will produce a good profile.

Using Quantifiler results• If final quantity is 1ng/µl then by simply adding 1µl one

will have the necessary amount of target DNA.

• What if value is 4.25 ng/µl.• For every 1µl there is 4.25 ng of DNA. So 1µl of extract

will be greater than optimum input level for the amplification kit. This sample needs to be diluted before it can be amplified.

• Calculation

• 4.25ng = 1µl • 1ng = 1 / 4.25 = 0.235µl

Using Quantifiler results• Calculation

0.235 µl of a sample is too little to be added. Pipetting error is common when using such low quantities of sample. By diluting the sample, more of diluted sample can be added and pipetting errors can be reduced.

Dilution by a factor of 10

1 µl of extract to 9µl of TE buffer.

After dilution by a factor of 10 simply multiply volume by 10

0.235µl x 10 = 2.35 µl

So for a quantity of 4.25ng / µl. It should be diluted by a factor of 10 and 2.35 µl of diluted sample be added to amplification tube.

Using Quantifiler results• At end of quantitation the quantity of DNA is 0.365ng/µl.• So 1µl of sample will not give 1ng of DNA.• To calculate how much µl will be needed to give 1ng of

DNA.

• 0.365 ng = 1µl• 1 ng = 1/ 0.365 = 2.73 µl• So you will need to add 2.73 µl of extract to get 1 ng of

DNA for amplification.

Using Quantifiler results• If you are using a 25 µl reaction for the STR amplification

then the amount of sample being added is limited.

• If the quantity of DNA is too low then it will require more than the amount of DNA that can be added to the reaction.

• In these cases it is advisable to concentrate DNA and quantitate again before going on to amplification.

Using Quantifiler results• At the end of this process samples should be

ready for STR amplification.

As long as the quantifiler results show there is enough DNA that the amplification kits require to generate a full profile then the analyst should be confident they will be able generate a full profile at end of STR analysis.

Using Quantifiler results• One important limitation to consider is if there is

a mixture of DNA in the sample the quantity generated is how much total DNA is present.

• The resulting profiles may be two or more incomplete profiles or one full profile and one incomplete profile.

• Total Human quantitation cannot tell if the final value is from more than one person.