1 Quantitative Real-time PCR (qPCR) Presented by Solmaz Oskooei 2 - - PowerPoint PPT Presentation

1

2

Quantitative Real-time PCR (qPCR)

Presented by Solmaz Oskooei

Jenny Carter, Kamila Jagiello, Rubin Wang

3

What is qPCR? What do we use it for at GOSH? How does it work? What can go wrong? Recent Cases NGS follow up

Introduction

4

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).

Introduction

5

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

Introduction

6

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!)

Introduction

7

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.

Introduction

8

What is the difference between QF-PCR (quantitative fluorescence PCR) and qPCR?

Introduction

qPCR

• Non labelled primers
• Real time measurements
• f quantity
• “Bespoke” primer

design QF-PCR

• Labelled primers
• Utilises STRs
• Can include several

probes in one assay Both can be used to determine copy number

9

Neither of these are the same as qPCR………

Introduction

qRT-PCR Quantitative reverse- transcriptase PCR RT-PCR

• Reverse transcriptase PCR

10

Primer design

Primer Design

Primer Testing Reporting qPCR Analysis/ 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!)

11

Primer Testing

• 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

Primer design Primer Testing Reporting qPCR Analysis/ Checking

12

Primer Testing

Blank (NTC)

13

Setting Up a qPCR

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

Primer design Primer Testing Reporting qPCR Analysis/ Checking

14

Setting Up a qPCR

• 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.

Primer design Primer Testing Reporting qPCR Analysis/ Checking

15

Analysis

16

Analysis

Relative Quantification

• Compare the RQ (Relative Quantification) value for each patient against a

normal control

Primer design Primer Testing Reporting qPCR Analysis/ Checking

RQ range Normal 0.8 - 1.2 Gain >1.3 Loss <0.7 Inconclusive 0.71 - 0.79 or 1.21-1.29

17

Analysis

Proband Mother Control1 Control2 Proband Mother Father Control1 Control2

Inherited gain Inherited loss

18

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

19

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.

20

What can go wrong?

Problem Solution

Primer design problems: e.g. SNPs, amplification in blank wells, haplotype, pseudogenes

• Re-design and repeat with new primers.
• Use SNP-free controls (not available for

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!

Inconclusive result

repeat Variation in normal population Do not follow up DNA degradation Request another sample Repeat so much that run out of DNA Request another sample

21

• 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!

What can go wrong?

22

Case 1

Region of LOH Homozygous deletion

arr 5q33.1(151,232,711-151,555,856)x0

• Includes first six exons of GLRA1 gene
• Homozygous deletions associated with hyperekplexia

23

Cases

qPCR follow up proband and parents: Father is normal!!?? Proband Mother Father

24

Case 1

Region of LOH Homozygous deletion Likely maternal segmental UPD

25

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

26

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?

27

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.

28

Case 3

Proband Mother Father Control1 Control2 arr 15q11.2(22,770,422-23,214,340)x1 pat NIPA1 gene – neurosusceptibility syndrome

NGS follow up cases

• 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

NGS follow up referrals Sep 2015 – June 2016 Total 19 Average TAT 44

1 2 3 4 5 6 7 8 9 10 NGS result confirmed NGS result not confirmed qPCR not possible Inconclusive

30

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:

• internal qPCR OMNI report

Micorarray follow up:

• proband array report updated,
• parental reports issued on

OMNI

31

32

33

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

34

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!

35

ANY QUESTIONS ?