high throughput qpcr: tips for analysis across multiple plates

Dr Mikael KubistaFounder and CEO, TATAA Biocenter

Presented by:

High throughput qPCR: tips for analysis across multiple plates

qPCR by sales people is VERY SIMPLE!

Compare to reference sample! Compare to reference gene!

Challenges in high throughput expression profiling

• The number of reactions does not fit into a single plate

• Number of target molecules per aliquot varies due to low numbers

• Testing for genomic DNA background by performing RT-controls are prohibitively expensive or not feasible

• Testing RNA integrity using microfluidics is prohibitively expensive

• Data analysis using methods such as t-test is unreliable due to multiple testing ambiguity

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Cq: 25.5 29 32.5

ΔCq= 29 – 25.5 = 3.5

ΔCq= 32.5 – 25.5 = 7

ΔCq= 32.5 – 29 = 3.5

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Cq: 27 30.5 34

ΔCq= 30.5 – 27 = 3.5

ΔCq= 34 – 27 = 7

ΔCq= 34 – 30.5 = 3.5

Cq’s depend on threshold. ΔCq’s don’t.

GOI RGS1 24 22S2 31 23

Compare relative expression in two samples

Calculate ΔCq

= 24-31 = 22-23

= 24-22

= 31-23

There are two ΔCq’s!

GOI RG ΔCqS1 24 22 2S2 31 23 8ΔCq -‐7 -‐1

Calculate ΔΔCq

= (24-31) – (22-23)

= (24-22) – (31-23)

GOI RG ΔCqS1 24 22 2 ΔΔCqS2 31 23 8 -‐6ΔCq -‐7 -‐1

ΔΔCq -‐6

Calculate Relative Quantity

GOI RG ΔCqS1 24 22 2 ΔΔCqS2 31 23 8 -‐6ΔCq -‐7 -‐1 RQ 64

ΔΔCq -‐6 64 CqΔΔ−= 2

Breaking up a large study into several plates

16 × 24 = 384 reactions

384/96 = 4 plates

”All Samples”

Plate 1 Plate 2 Plate 3 Plate 4

Samples held together (”All Samples” layout)

offsetPlate 1 +1Plate 2 +2

= ((24+1)-(31+1)) – ((22+2)-(23+2))

GOI RG ΔCqS1 25=24+1 24=22+2 1 ΔΔCqS2 32=31+1 25=23+2 7 -‐6ΔCq -‐7 -‐1 RQ 64

ΔΔCq -‐6 64

In real the offsets are not known.Here we assign arbitrary numbers to trace there impact only.

”All Genes”

Plate 1

Plate 2

Plate 3

Plate 4

Genes held together (”All Genes” layout)


= ((24+1)-(31+2)) – ((22+1)-(23+2))


ΔΔCq -‐6 64

”Mixed layout”

Plate 1 Plate 2

Plate 3 Plate 4

“Mixed layout” with two genes and two samples


= ((24+2)-(31+1)) – ((22+1)-(23+2))


ΔΔCq -‐4 16

The Inter-Plate Calibrator (IPC)

GOI RGIPC 20 21


= (((24+2)-(20+2))-((31+1)-(20+1))) – (((22+1)-(21+1))-((23+2)-(21+2)))

GOI RGS1 26=24+2 23=22+1S2 32=31+1 25=23+2

IPC_A 21=20+1 22=21+1IPC_B 22=20+2 23=21+2ΔCq -‐6 -‐2ΔΔCq -‐7 -‐1 RQ

ΔΔΔCq -‐6 64

Relative quantification on multiple plates

When expression is normalized to reference genes and samples are compared (ΔΔCq) multiple runs can be merged for common analysis without correction if either:

• All genes for all sample are measured together in the same plate (“All genes”)

or

• All samples for all genes are measured together in the same plate (“All samples”)

Interplate calibrator

• Interplate calibrators are used to compensate for variations between runs due to instrument settings (base-line correction and threshold settings)

• Interplate variation depends on the instrument channel used, but is virtually independent of assay.

It is highly discouraged to perform independent inter-plate calibrations per assay!

• The Cq of an interplate calibrator must be measured with very high accuracy, else interplate calibration may add more variance to the data than the systematic variation it removes.

• Interplate calibrators should be:– Very stable assays – Uncomplicated, purified template at fairly high concentration (20 <Cq < 25)– Run in replicates (minimum triplicates)– The Interplate calibrator shall be stable over time

www.tataa.com/products-page/quality-assessment/tataa-interplate-calibrator/

How many preamplification cycles?

Average number of targets per reaction container should be 35 for accurate analysis.

If we assay for 100 targets the original sample should have

3500 of each.

FACSAspirationCapture


Cellulyser

No losses!

Freezes profile!

Cell’s expression changes in matter of seconds in response to environmental changes


Cellulyser GrandScript

EfficientRT

Anders Ståhlberg, Mikael Kubista, and Michael PfafflComparison of Reverse Transcriptases in Gene Expression AnalysisClinical Chemistry 50, No. 9, 2004


Cellulyser GrandScript GrandMasterPreAmp

Efficient Preamp

Highly optimized assays

Ø Dynamic rangeØ SensitivityØ Specificity

gBlocks® Gene Fragments


Cellulyser GrandScript GrandMasterPreAmp

High throughputqPCR

GenEx iReport

Compensate for gDNA background: the ValidPrime

+ gDNA specific assay (ValidPrime)+ Reference gDNAOriginal data gene 1 gene 2 gene 3 gene 4 ValidPrime

sample 1 20.1 31.1 22.1 28.2 32.5sample 2 20.5 31.2 22.5 28.9 33.2sample 3 21 31.1 22.9 30.2 32.3sample 4 23.1 31.8 22.5 32.3 34.2sample 5 23.5 30.8 22.8 32 33.1

gDNAstandard 25.8 26.9 26.7 26 27

Laurell et al., Nucleic Acids Research, 2012, 1–10;; Drug Discovery World (2011)

( )ValidPrimegDNA

GOIgDNA

ValidPrimeSample

GOIRT CqCqCqCq −+=−

More accurate and more cost effective than RT(-) controls

•15% of human genes have pseudo genes• Pseudo genes usually lack introns

• Pseudo genes are often present in multiple copies

Calibrated against NIST SRM2372

Human genomic DNA

Traditionally RNA integrity is tested by electrophoresis

RNA extracted from liver tissue. Left at room temperature and analyzed (Bioanalyzer/Experion/Fragment Analyzer)

0min -------------------------------------------------->120min

Works quite well, but way too expensive for high throughput applications!

Molecular approach: ΔAmp and the ERR

Differential amplicons (ΔAmp)

Target

Short (S)Medium (M)

Physical/chemical Degradation

Björkman et al., Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity. BiomolecularDetectionand Quantification2015.

Enzymatic Degradation

Endogeneous RnaseResistant (ERR) marker

Stability marker

Not detected by

electrophoresis

RNA degradation by formalin detected with ΔAmp

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P P IA - E R R m a rk e r (P ie c e s )

R Q I (P ow d e r )

P P IA - E R R m a rk e r (P ow d e r )

Activation of astrocytes in response to trauma

Astrocytes(principal role in repair)

Single cell expression profiling FACS sorted astrocytes from mouse brain Response to trauma (focal cerebral ischemia)

Comparing genes one by one Gene P-‐ValueAqp9 1.00E-‐08gene 1.00E-‐08gene 1.00E-‐08gene 1.00E-‐08Grin2a 1.00E-‐08Grin2d 1.00E-‐08Grin3 1.00E-‐08Kcna3 1.00E-‐08Snap 1.00E-‐08Gluk1 1.26E-‐07Pdgfr 1.79E-‐06Glun3a 2.78E-‐06Cspg4 4.13E-‐06Vim 8.18E-‐06Kcnk2 3.57E-‐05Gfap 9.98E-‐05Gluk3 0.000416Grin1 0.000867S100b 0.003769Kcnj10 0.004225Gria1 0.012991Kcna5 0.025924Grin2b 0.030311

Approach suffers from multiple testing

ambiguity and low power and does not exploit

correlation

3D PCA classification of single astrocytes – all genes

QC products from TATAA

Gene panels• Truly Stem Validated primers for 13 markers for stem cell differentiation• CTC GrandPerformance panel for circulating tumor cells

CelluLyser Lysis and cDNA Synthesis Kit• CelluLyser For single cell lysis

Quality control

• ValidPrime to test the quality of analyzed mRNA in complex samples• Exogenous controls DNA and RNA spikes to estimate yields and test for inhibition• InterPlate calibrator kit to remove variation between runs• DAMP and ERR to test RNA integrity

Software• GenEx for qPCR data mining

Training modules from TATAA

1 day qPCR for miRNA analysis

1 day Sample preparation and quality control

1 day Genotyping with qPCR

1 day Immuno-qPCR

1 day Multiplex PCR

1 day Quality control of qPCR in MDx

1 day CEN/ISO guidelines for the preanalytical process in MDx

2 days Hands-on qPCR

2 days Single cell analysis

2 days Experimental design and statistical data analysis

2 days Digital PCR –Applications and analyiss2 days NGS – Library construction and quality control

3 days Experimental design and statistical data analysis

3 days Hands-on qPCR

Specifications for pre-examination processes

• FFPE tissue — RNA• FFPE tissue — DNA• FFPE tissue — Extracted proteins• Snap frozen tissue — RNA• Snap frozen tissue — Extracted proteins• Urine, plasma, serum: Metabolites• Blood — Circulating cell free DNA• Blood — Genomic DNA• Blood — Cellular RNA

http://www.tataa.com/courses/

gene expression

PrimeTime® qPCR Assays• Primer and probe sequences provided• Free design tools• Available predesigned for human, mouse, and rat

www.idtdna.com/primetime

Thank you!

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high throughput qpcr: tips for analysis across multiple plates

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