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Quantitative Real-time PCR (qPCR) Presented by Solmaz Oskooei 2 Jenny Carter, Kamila Jagiello, Rubin Wang
Introduction What is qPCR? What do we use it for at GOSH? How does it work? What can go wrong? Recent Cases NGS follow up 3
Introduction What is qPCR? Quantitative real-time PCR Measurements of fluorescence are made in real time during the exponential phase Determines quantity of an amplified product (copy number). 4
Introduction What do we use qPCR for in the laboratory? 1. To confirm a microarray result (gain or loss of copy number) 2. To determine inheritance (testing parents) 3. Further investigation of a potential copy number change found via NGS 4. NIPD – fetal sexing 5
Introduction How does it work? Design primers for a ~100bp region of interest Measure patient and control DNA to ensure all at the same starting concentration and amount. Add primers and Master mix including SYBR Green. (can use Taqman but SYBR green is cheaper and faster!) 6
Introduction How does it work? • PCR takes place along with melt curve analysis all in one reaction • Measurements of fluorescence are taken in real time. • Amount of amplified product from patient is compared to control = relative quantitation. • Results normalised using endogenous controls (VCPIP1 and MANEA) • A stunning bar graph is produced. 7
Introduction What is the difference between QF-PCR (quantitative fluorescence PCR) and qPCR? qPCR QF-PCR Both can be -Labelled primers used to -Non labelled primers determine copy -Utilises STRs number -Real time measurements of quantity -Can include several probes in one assay - “Bespoke” primer design 8
Introduction Neither of these are the same as qPCR……… qRT-PCR RT-PCR Quantitative reverse- -Reverse transcriptase PCR transcriptase PCR 9
Primer Design Primer Primer Analysis/ Reporting qPCR design Testing Checking Good design is crucial as qPCR is VERY sensitive. Use several resources : - Alamut - ENSEMBL - Primer-BLAST - gnomAD - OligoCalc - UCSC insilico PCR 95-125bp amplicon Two pairs for every family Details entered onto primer database (1870 primers so far!) 10
Primer Testing Primer Primer Analysis/ Reporting qPCR design Testing Checking All new primers are tested by running a qPCR with and without control DNA. This is to check for primer specificity. Can test 16 primer pairs per plate Discarded if fail primer testing 11
Primer Testing Blank (NTC) 12
Setting Up a qPCR Primer Primer Analysis/ Reporting qPCR design Testing Checking Once a suitable primer has been identified for a family, a worksheet is produced. One 96 well plate will contain: Two families (total of up to 6 patients) Two normal DNA control samples (one male, one female) Two control primers (endogenous controls MANEA and VCPIP1) Blank (NTC) control wells for all 4 primers 13
Setting Up a qPCR Primer Primer Analysis/ Reporting qPCR design Testing Checking 1. DNA Quantification Getting the same starting DNA concentration is critical!! 300ng DNA per patient 2. Primer Preparation Primers are added to tubes containing the Power SYBR green master mix. 3. Loading DNA + Primer Then divide each DNA/primer mix between three wells on 96 well plate. 14
Analysis 15
Analysis Primer Primer Analysis/ Reporting qPCR design Testing Checking Relative Quantification Compare the RQ (Relative Quantification) value for each patient against a normal control RQ range Normal 0.8 - 1.2 Gain >1.3 Loss <0.7 Inconclusive 0.71 - 0.79 or 1.21-1.29 16
Analysis Proband Mother Control1 Control2 Inherited gain Proband Mother Father Control1 Control2 Inherited loss 17
Advantages Can be used to detect any copy number changes (depending on location), down to a minimum of 100bp. Relatively inexpensive Primers can be designed and ordered relatively quickly and easily Targeted test – avoids incidental findings 18
Limitations May not be possible to design a primer Preparation and set up is labour intensive (2-3 hours) Low throughput as can only do two families per plate, maximum of two plates per day, per machine. No positional information for duplications. Very sensitive – technically challenging, precise pipetting essential. Cannot detect mosaicism Cannot determine size of copy number loss Does not give an integer copy number value. 19
What can go wrong? Problem Solution • Primer design problems : e.g. SNPs, Re-design and repeat with new primers. • amplification in blank wells, haplotype, Use SNP-free controls (not available for pseudogenes microarray follow ups) • use two sub-optimal primers together • use a different technique (FISH/array) Contamination in blank wells repeat and discard working solutions High SD repeat Discrepant controls discard controls and repeat with different controls. Check to make sure ROI is not in a variable region! repeat Inconclusive result Variation in normal population Do not follow up DNA degradation Request another sample Repeat so much that run out of DNA Request another sample 20
What can go wrong? Therefore even if a primer has worked well previously – it still may take several attempts at setting up to get a result! Repeating multiple times can ramp up costs Design and receipt of primers can take time especially if re-designs required This is why we sometimes have long TATs This is also why we are unable to test multiple primers for one copy number change…..…. it could take up to a year to get a result if we did that! 21
Case 1 Homozygous deletion Region of LOH arr 5q33.1(151,232,711-151,555,856)x0 • Includes first six exons of GLRA1 gene 22 • Homozygous deletions associated with hyperekplexia
Cases qPCR follow up proband and parents: Proband Mother Father 23 Father is normal!!??
Case 1 Homozygous deletion Region of LOH Likely maternal segmental UPD 24
Case 2 BBS9 Bardet-Biedel Syndrome From the NGS panel, several patients appeared to have a homozygous deletion within this gene qPCR was consistent with this finding 25
Case 2 BBS9 However, when qPCR was repeated for an affected sibling and parent, controls appeared to have deletions! qPCR repeated 6 times, with different controls - some controls appeared homozygous, some heterozygous, some normal. We could not issue a report and recorded the qPCR as inconclusive. The controls are parents of micorarray probands who are normal via qPCR for the amplicon tested at the time (they have not been arrayed or sequenced). What was going on? 26
Case 2 BBS9 A SNP issue? No. The primers included SNP regions but all of the BBS9 patients were SNP free. Beth did a lot of investigating and found that a mobile insertion element had been seen in the 1000 genomes pilot project. This is recorded on the DGV (Database of Genomic Variants). Worth bearing in mind that some results could be caused by normal population variation. 27
Case 3 Proband Mother Father Control1 Control2 arr 15q11.2(22,770,422-23,214,340)x1 pat NIPA1 gene – neurosusceptibility syndrome 28
NGS follow up cases 10 9 8 NGS follow up referrals 7 Sep 2015 – June 2016 6 5 Total 19 4 3 Average TAT 44 2 1 0 NGS result NGS result not qPCR not Inconclusive confirmed confirmed possible qPCR provides more evidence on whether or not the NGS result reflects a real copy number change We design primers only within exons We test two sets of primers from within the exon(s) of interest but cannot use multiple primers to try to work out the exact size of the copy number change
Workflow Cyto duty scientist receives microarray follow up referral OR molecular scientist emails Cyto qPCR team Senior scientist activates for qPCR on our LIMS qPCR Enter result on OMNI NGS/other follow up: Micorarray follow up: - internal qPCR OMNI report - proband array report updated, - parental reports issued on OMNI 30
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Summary qPCR is great for detecting specific copy number changes Relatively cheap Can be used for copy number changes down to a minimum of 100bp Freedom to design primers anywhere in the genome But Repeats are often necessary so TATs often go over 28 days. If we have tested a family member previously, it doesn’t necessarily mean that testing another will be quick! qPCR can’t tell you the extent of the copy number change We can’t test multiple primer pairs for one copy number change 33
CONCLUSION We will continue to use it particularly for NGS follow ups as these often have single exon deletions which are difficult to confirm any other way. It is considerable cheaper than running an array. For each patient cost excluding labour is approx. £250 for an array compared to around £25 for qPCR. Success rate is also good – we rarely fail cases. The usual problem is that we run out of DNA after all of the NGS work and then have to ask for a new sample! 34
ANY QUESTIONS ? 35
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