Quantitative PCR Session 3: Instrument and Software Presented by: Robert O'Brien Training Specialist – Forensic Biology

Quantitative PCR Session 3- Instrument and Software Instrument Operation AB 7500 Processing Multicomponent data Initial Instrument Set up Software Set-up

Instrument Operation AB 7500

Instrument Operation Basic Principle Light hits a tube/ vessel containing the PCR reaction once per cycle Fluorescent dyes if activated emit light corresponding to their spectral characteristics Emitted light is focused on a detector Computer software interface interprets the detector signal

Instrument Operation For AB 7500 light source is a tungsten halogen lamp Fluorescent dyes have wavelengths in 500nm to 700nm range Filters are in place which allow light of a specific wavelength onto the detector The detector is a CCD camera (CCD- charged couple device).

Instrument Operation CCD- Charged couple device is a light sensitive integrated circuit that stores and displays data for an image in such a way that each pixel in the image is converted into an electrical signal. The electrical signal is read by the software

Instrument Operation The AB 7500 has a thermal block which is needed for the heating and cooling that is required for the PCR reaction The filter the light passes through allows for multiplexing of different dyes as mentioned earlier FAM dye for DNA sample, VIC dye for IPC

Instrument Operation

PCR detection system Tungsten halogen lamp directs light to each well of a 96 well optical plate Light passes through the optical adhesive cover and excites the fluorescent dyes in the samples A system of lenses, filters, and a dichroic mirror focuses the fluorescence emission into a charge- coupled device (CCD) camera. Based on the wavelength the filters separate the light into a predictably spaced pattern across the CCD camera

Instrument Operation

PCR Detection system During the run the CCD camera detects wavelengths between 500nm and 660nm from each well The SDS (Sequence Detection Systems) software obtains the fluorescence emission data from the CCD camera and applies data analysis algorithms

Instrument Operation

Processing multicomponent data During the multicomponent transformation the SDS software uses algorithms to determine the contribution of each dye. Before the contribution of the individual dyes are assessed other contributions to the composite spectrum of the dyes must be taken into account.

Quantitative PCR

Processing multicomponent data An algorithm removes the background component stored in the background calibration file A background calibration run is done when the instrument is first set up. The background calibration run determines how much background signal the instrument produces during a quantifiler run.

Processing multicomponent data Pure dye standards are run during initial instrument set up The software is able to use the extracted pure dye standards to express the composite spectrum in terms of the pure dye components Once the composite spectrum is broken down into its pure dye components an algorithm applies matrix calculations to determine the contributions of each dye component

Processing multicomponent data Measured spectrum = a(FAM) + b(VIC) + c(ROX)+ d(Background) + MSE MSE- Mean standard error- This indicates how closely the collective multicomponent spectrum conforms to the raw spectra

Processing multicomponent data Purpose of ROX ROX- a passive reference dye It is not involved in the PCR reaction It is used to normalize the reporter signal

Processing multicomponent data ROX reporter dye is present in every well so it is exposed to the same conditions as the samples in the well Therefore if there are fluctuations in the signals produced by the dyes that may be related to electrical fluctuations for example, the ROX dye will also experience these fluctuations which are taken into account when interpreting the strength of the signal and in turn the quantity of DNA.

Processing multicomponent data The SDS software normalizes each reporter signal by dividing it by the fluorescent signal of the passive reference Rn (normalized reporter signal)= Reporter fluorescence Passive reporter fluorescence Normalizing the reporter signal allows the software to make better well to well comparisons of reporter signal During real-time PCR the Rn is increasing as the amount of specific amplified product increases

Processing multicomponent data Background – is the fluorescence given off during the first few cycles of the PCR reaction. Baseline is set during the first few cycles usually between 6 and 15 Software then calculates the ΔRn ΔRn = Rn value – baseline Cycle where ΔRn value crosses the threshold is the C T value.

Initial Instrument Set-up When the AB 7500 first arrives in the lab several calibrations must be done before the instrument can be used to run samples Region of Interest calibration (ROI) Background calibration Pure Dye Calibrations Instrument verification run

Initial Instrument Set-up Even though only a few people will have to be involved in the initial instrument set-up it is important that everyone knows what occurs during this process. Also the same calibrations performed during set up also need to be done as part of the regular maintenance.

Initial Instrument Set-up Region of Interest calibration (ROI) ROI – This is mapping the positions of the wells on the sample block so that the software can associate increases in fluorescence with different wells The instrument uses a set of optic filters to distinguish the fluorescence emissions gathered during the runs ROI generates a calibration image for each individual filter to account for minor differences in the optical path

Initial Instrument Set-up Region of Interest calibration (ROI) It is expected there will be minor fluctuations throughout a plate from well to well The instrument must know what these fluctuations are so they are not included in what the instrument is reading as fluorescence from the samples in the wells ROI normalizes the wells Since you are dealing with very small amounts of DNA minor fluctuations can change final quantity readings

Initial Instrument Set-up Background calibration Purpose of background calibration is to measure the amount of background fluorescence in the instrument The measure of the background fluorescence is important so in future runs this value can be subtracted out of the fluorescent signals from the samples Sources of background fluorescence Background electronic signal Contaminants in sample block Plastic consumables (plates and caps)

Initial Instrument Set-up Background calibration If contamination is detected in the sample block then the wells where the contamination was identified should be cleaned and the background calibration run again. During runs it is very important not to introduce any new contamination. This is why it is important that the base of optical plate is not handled or does not touch the counter surface. Contamination from handling may be incorrectly recorded as low levels of DNA in blank sample wells.

Initial Instrument Set-up Pure Dye Calibrations Purpose- to provide the software with a way to distinguish the fluorescent signals from each dye This is important because during a run the fluorescence from all the dyes come off together in a collective fluorescence The software must have a way to separate out the individual contributions of the dyes in order to interpret their signal and the resulting quantities that will be calculated based on their fluorescence

Initial Instrument Set-up Pure Dye Calibrations Pure dye calibration- collects spectral data from a series of pure dye standards. It then stores the spectral information in the pure spectra run file which is a calibration file in the SDS directory. Software can then use the pure spectra data during subsequent runs to characterize pure dyes and distinguish the individual contribution of each dye in the collective fluorescence.

Initial Instrument Set-up Pure Dye Calibrations A pure dye calibration is done for each dye in a separate plate. The pure dye plates are supplied by Applied Biosystems and they include all the dyes that are used for sample and IPC detection during a quantifiler run.

Initial Instrument Set-up Instrument verification run Purpose- To test the system after all the calibrations have been performed and before samples and standards are manually set up This run uses a RNase P plate which is preloaded with the reagents necessary for the detection and quantitation of genomic copies of the human RNase P gene RNase P gene is a single copy gene encoding the RNase moiety of the RNase P enzyme

Initial Instrument Set-up Instrument verification run Each well of the plate used for instrument verification contains : 1x Taqman Universal PCR Master Mix RNase P primers FAM dye labeled probe Known concentrations of human genomic DNA template All these components are preloaded in the plate so any problems with the run will be due to the instrument and not due to plate set up which can happen if plate was set up in house.

Initial Instrument Set-up Instrument verification run After the run is complete the SDS software generates a standard curve from the averaged cycle (C T ) values of the replicate groups of standards The SDS software then calculates the concentrations of two unknown sets of samples using the standard curve. Then it calculates the mean quantity and standard deviation for the different unknown sample populations to assess the instrument’s performance.

Initial Instrument Set-up Instrument verification run The instrument passes the verification if the analyzed data demonstrates that the instrument distinguishes between the unknown genome equivalents with 99.7% confidence level.

Software set- up The software must be set up before the instrument can be used to run any samples The software used in quantifiler is SDS software – SDS- Sequence Detection System This set up should only need to be done one time and then the information saved so it can be used for future runs The following must be set up Plate documents Thermal cycler conditions

Software set- up Plate Documents A Plate document is a representation of the arrangement of samples (which includes standards and unknowns) and detectors on the reaction plate Plate documents are created using the SDS software

Software set- up Plate Documents Two types of plate documents 1)SDS document-.sds – This is the primary file to use when performing a run. Required for all experiments 2)SDS template-.sdt- File that already contains run parameters that are commonly used in plate documents, such as detectors and thermal cycler conditions

Software set- up Plate Documents A Plate document must be set up so that it corresponds exactly to the arrangement of the standards and unknown samples in the reaction plate.

Software set- up Plate Documents Setting up a Plate document To create a new plate document AB Prism 7500 Go to AB Prism 7500 SDS Software Once it is opened then go to File New The next step will be to create detectors. This is only done the first time the plate document is created.

Software set- up Plate Documents Tools- Detector manager- File- New At this point detectors will be created for Human and Y plates Both Human and Y kits use FAM as the reporter dye After the information is put in then click on “Create Another” to save and set up a new detector

Software set- up Plate Documents For Human and Y detectors the name must be entered The dye selected is the same FAM Description can be left blank Quencher dye is left at default none Color is selected to represent FAM Same color can be chosen for both since one sample will not have both human and Y in same well

Software set- up Plate Documents

Software set- up Plate Documents After Human and Y detectors are set up then a detector is set up for the IPC The IPC reporter dye is VIC not FAM Quencher dye is left at the default (none) Color chosen to represent IPC is different from the color chosen for FAM At the end of this click OK to add the IPC detector and to return to Detector manager dialog box

Software set- up Plate Documents

Software set- up Plate Documents At the end of the set up the detectors should look like the picture below Notice only one detector either Human or Y is chosen per well

Software set- up Plate Documents Once the detectors are created they can be added to the Plate document Under Tools go to Detector manager then select Quantifiler Human, Y and IPC detectors by clicking them while pressing CTRL key Then click ADD to Plate Document Click Done

Software set- up Plate Documents Now that the detectors have been set up they must now be applied to all the standards View- Well Inspector- Then select the wells that the standards will be put into. Most laboratories will always put the standards in the same wells on the plate Also all standards are run in duplicate so the same information must be entered for both wells corresponding to the standards

Software set- up Plate Documents Select Wells used for first standard by holding down CTRL while selecting them Open the well with Well inspector under Views First name the Standard Either numbers 1-8 or letters A-H are generally used Then under Task menu use drop down menu to select Standard Then under quantity put in the quantity of the standard No units are entered but it is assumed to be ng/µl

Software set- up Plate Documents Standards are done for Human and Y wells independently so under Task only select Standard and put in quantity for Human or Y not both in same well A completed STD well

Software set- up Plate Documents A completed plate for Human only should like the following. A duplicate set of standards and all the wells labeled

Software set- up Plate Documents Setting detectors for sample wells Under Plate tab – select wells that correspond to where unknown samples will be With wells selected click on View then Well Inspector Then depending on whether the sample is for Human or Y click the appropriate box under USE Whether it is Human or Y IPC should always be checked under USE After this is done then in the individual wells put in the names of the samples

Software set- up Plate Documents Example of a completed Well Inspector for a sample

Software set- up Plate Documents

Software set- up Thermal cycler conditions Before using the AB 7500 two changes must be made to the thermal cycler conditions. The Thermal profile and sample volume must be adjusted Thermal Profile- First stage in thermal profile the HOLD stage must be deleted Sample volume should be changed to 25µl and the 9600 Emulation should be checked Checking 9600 emulation box reduces the amp rate

Software set- up Thermalcycler conditions Instrument tab Press shift key and click on stage 1 which is the HOLD stage After HOLD step is selected press the delete key After this is done the first stage should be 1 Rep at 95ºC for 10 mins This is the same step in hot start PCR which is used to activate the Ampli Taq Gold DNA polymerase

Software set- up Thermal cycler conditions

Software set- up After the Plate document is completed it is saved as a.sds file FILE- SAVE (select location where it will be saved) Enter a file name save as type select SDS document.sds Click save

Software set- up Template files are generic Plate documents that are set up to ensure uniformity in the laboratory. Most laboratories will set up template documents so that everyone is using the same Plate document when running their quantifiler. The only information that would be changed from one plate document to the next would be the names of the samples and whether they are for Total Human or Y quantitation.

Software set- up Template Documents contain the following information –Assay- specific detectors –Well assignments for quantification standards with detectors, tasks and quantity –Well assignments for unknown samples with detectors already set so only the appropriate assay needs to be checked –Instrument settings to include thermal cycler and reaction volume settings Template documents are saved as a.sdt file and saved under templates

Software set- up

A completed Well inspector in a template file