Bio-Reference

Standards/Materials

Carole Foy

LGC

Presentation to EuroGentest

15th May 2007

Overview of Presentation

• Role of Horizontal Standards in International Bio-Metrology

• Bio-reference standards developed at LGC

• Lessons learnt in production/use of materials

Vertical cf. Horizontal Standardisation

• Vertical reference standards – product/gene/matrix specific e.g. Cystic Fibrosis reference panels

• Horizontal reference standards - generic, link to method performance criteria

Te

ch

niq

ue

• Calibrate/validate method, operator performance, comparability of different technology platforms….

Product A B C D

Real-time PCR

Microarray analysis

PCR-RFLP

Sequencing

Bio-Reference Materials at LGC

• Microarray QC Material

• Real-time PCR QC Material (collaboration with NIST, plasmid based)

• Real-time PCR QC Kit Material

• Genotyping/haplotyping QC material

• “Biomarker” panel – multiple RNA transcripts (2007 - )

Additional Bio-reference material activities

• UNIQ PT scheme launched, with 2 rounds being carried out

Reference material production and distribution:

• ATCC products

– Cell lines and DNA

• Gentris

– GentriSure Human Genomic Reference Controls (Pharmacogenetics)

Specificity Standards and Performance

Indicators for microarrays

• Assist in assay optimisation, QC, validation and comparability

• Multiple sets of standards (high, med, low GC content)

• Four standards within each set differing by 1,3 and 5 mismatches

• Fluorescently labelled reverse complements (Cy3 and Cy5) spiked-in

• Sub-array profiles compared to “average” profile for slide QC

• Discriminatory power of each panel assessed

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Probe Standard

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Plasmid Based Performance Standard for

qPCR

• Designed as a independent monitor of equipment and technical staff/lab performance

• Ct values by real-time PCR within a defined range • A plasmid with a synthesised segment that is not homologous to

any known gene• Sequence suitable for most probe types

Ct

valu

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Log target amount

Real-time PCR Kit QC Material

• Designed for laboratories wishing to check the

consistency of their real-time PCR measurements over

time

• Kit consists of:

– Forward Primer (21mer oligonucleotide)

– Reverse Primer (20mer oligonucleotide)

– Target (86mer oligonucleotide)

– Probe (24mer)

Genotyping/haplotyping QC material

• Genotype/haplotype-validated reference material

– platform evaluation

– analyst training

– assay QC

– trace DNA detection (e.g. circulating cancer and fetal markers)

– rapid DNA detection (e.g. Point Of Care Diagnostics)

• Non-human (Arabidopsis)

– Two strains selected which differ by 12 SNPs in ~1500bp fragment

– Fragments cloned and amplified

• Poly A tail and T7 promoter added to allow IVT for gene expression

“Biomarker” Reference Panel

• Increased volume of healthcare information available

– Many potential new biomarkers

• Increased complexity of healthcare information

– Panels of biomarkers rather than a single analyte

• Multi-analyte analysis

– Overall trends and patterns

– Video camera vs single click camera analogy

“Biomarker” material under development

(in collaboration with NIST)

• Calibrator sample for qRT-PCR multi-analyte

measurements covering a range of concentrations

• Used to normalise qRT-PCR measurements

• Used to evaluate dynamic range and precision of assays

• Based on ERCC standards

– Panel of 144 In-vitro transcripts

– Adopted by the array community as reference standards

UNIQ PT scheme

• Complementary activity to development of ref materials

• Designed to test laboratories ability to carry out real-time PCR measurements at a generic level

• Test samples are synthetic oligonucleotides cloned into a plasmid with no direct diagnostic relevance – No chance of contaminating in house processes

– No barriers to acceptance of materials

• Targets constructed at a range of GC contents

• Artificial matrix developed and tested

• Steering committee composed of experts from range of sectors

UNIQ PT Scheme

• 1 round successfully completed December 2006 – 13 labs (all UK/EU) returned results

– Results were generally good, but at a few labs had some problems with some measurements

– Feedback and technical help provided after the report to help labs identify/address potential issues

• 2nd round samples with 15 participants nearing completion– Some participants outside Europe

Lessons learnt ….

qPCR Plasmid Standard - Round 1 Pilot

Study

Assigned mean = 250.00 Consensus mean = 146.40

Graphs of equivalence - Round 1- Sample B

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qPCR Plasmid Standard - Round 2 Pilot

study

Assigned mean = 0.18 Consensus mean = 0.16

Graphs of equivalence - Round 2 - Sample B

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Improvement in performance between rounds

• Changes between rounds:

– Experience of participants (potential large effect)

– Form of target analyte (solution vs. dried-down DNA)

– Prescriptive protocol

– Submission of all raw data -negate potential bias

– Set number of replicates

– Data handling

– Verification of data collation

Relative errorCV

Round 2

Round 1

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Comparisons between rounds - values based on averages

Round 2

Round 1

Round 1 of UNIQ PT

• Participants requested to quantify a high concentration

DNA solution in house

• 12 unknown samples also distributed, with lower concentrations of the target

• 9 of the 12 unknowns required DNA extraction – 2 kits

recommended for use

• All 12 samples then analysed - quantification of target

DNA by Q-PCR

• Primer and probe for 5’-nuclease assay supplied

DNA extractionHigh concentration DNA quantification

Q-PCR quantification and reporting

Round 1 results

• Participants mainly used 2 methods for quantification of the high concentration DNA

– UV spectrophotometry (A260 measurement)

– PicoGreen kit (fluorescent intercalating dye)

• Participants were close to the expected ~20ng/ul

• Pico Green underestimated the concentration of DNA present by ~50%

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Laboratory number

DNA concentration ng/ul Measurements

using Pico Green

Variability of DNA quantification methods

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U-2000 ND-1000 PicoGreen Quant-iT

Platform

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English et al. 2006.

Use of elemental analysis to

determine comparative

performance of established DNA

quantification methods.

Anal Chem. 78(13):4630-3.

• Common DNA quantification methods can give different results on the same sample

• May be exacerbated by inappropriate standards and poorly calibrated equipment

DNA quantification results

• Each unknown supplied in triplicate

• qPCR amplification only

Median

Extraction and quantification

• More overall variability with the more complex analytical process

• Results coming in now for Round 2

Useful information on current practice

Use of method blanks

Yes

No

Separate DNA extraction area

Yes

No

Separate pre and post PCR

Yes

No

Calibrated pipettes

Yes

No

• Feedback on performance and possible problems also provided to participants if requested

Summary

• Need for inter lab comparisons of measurements and associated materials for use

• Horizontal reference materials can provide high level traceability of measurements for:-– calibration/validation of technique

– operator/lab performance

– comparability of different technology platforms

• Homogeneity and stability issues key for production of appropriate materials

Acknowledgements

• Nick Boley

• Lyndsey Birch

• Malcolm Burns

• Carol Donald

• Steve Ellison

• Claire English

• Jacquie Keer

• Helen Parkes

• Tim Wilkes

• Alison Woolford

• Marcia Holden (NIST)

• Marc Salit (NIST)

www.mfbprog.org.uk