Jack M. Gallup

PREXCEL-Q proof of principle and

the formation of the ISU qPCR

Consultation Service

PREXCEL-Q precepts:PREXCEL-Q precepts: Use “Stock I” (sample mixture) on a “Use “Stock I” (sample mixture) on a “Test Test

PlatePlate” to:” to:

1.) Determine the ‘inhibitory threshold’ (minimum 1.) Determine the ‘inhibitory threshold’ (minimum dilution required by samples to avoid qPCR dilution required by samples to avoid qPCR inhibition).inhibition).

2.) Identify the valid dilution range(s) of “Stock I” for 2.) Identify the valid dilution range(s) of “Stock I” for each target standard curve.each target standard curve.

3.) Determine the “Tier 1” dilution for each sample, 3.) Determine the “Tier 1” dilution for each sample, individually; per target (if necessary):individually; per target (if necessary):

4.) Determine if more than 1 “Tier dilution” is needed 4.) Determine if more than 1 “Tier dilution” is needed to accommodate rare or hyper-abundant targets. to accommodate rare or hyper-abundant targets.

“Tier 1” (the red-dot)

Typical Test Plate results exposing the “inhibitory characteristic” per target

LOG-linear ranges identified for each target

Best case scenario:

For simplicity’s sake, try to find the same “Homodynamic” range for all targets

0

5

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-8 -7 -6 -5 -4 -3 -2 -1 0

[log]

Once non-inhibitory sample dilutions are attained, the high-efficiency, LOG-linear amplification region becomes readily apparent …

• Primer and probe extinction coefficient-based dilution calculations (pre-qPCR)

• Processes all o.d.260nm sample readings• Automatic calculation of any dilution series• Spells out DNase treatments, RT reaction

and qPCR Master Mix set-ups• Avoids perfunctory math errors in the above• Let’s you know if a particular set-up is possible

or not (sample constraints etc.)• Calculates approximate price of each assay

What is it?

What does it do? How does it work? Is it easy to use?

Is it free? …Yes! …

Empirically:

It is a suite of 36 interwoven Excel 2003 files that automatically performs the common

calculations involved in qPCR - making theentire work-flow much less time-intensive.

• Spells out all sample and standard dilutions• Keeps track of over 65,000 samples• Assay Development/Project Management• Allows you to select the high efficiency regions

for each target of interest (and work within that)• Gives a “sample inhibition” report• Calculates safe extra into reagent prep. amounts• Generates an overall qPCR dynamic range report• Peace of mind (shows you where inhibition lurks)

df = (Upper/Lower)1/(p - 1)

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T

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Test Plate

Final Plates:

11 serial dilutions: from full-strength Stock I to 1:50,000 or 100,000

P-Q Cq analysis

Stock I used for standardsUnknowns diluted to respective Tier ng/uL spots

qPCR Consultation Service

http://vetmed.iastate.edu/isuqpcrconsultationservices

ISU qPCR Consultation Service For qPCR Assay Development and Project Management

 

In person and on-line consultation for comprehensive qPCR theory and design assistance from beginning to end. All steps discussed and printed out for immediate in-lab use.

General Services offered: 

Basic information/qPCR theory and math 

Primer-probe design assistance and suggestions 

Identifying appropriate reagents, master mixes and machine platforms for One- and Two-Step qPCR, including LCM-qPCR 

MIQE-based RNA isolation, DNAse treatment and reverse transcription reaction formulation suggestions and guidance 

Nucleic acid quality assessment and quantity measurement suggestions and guidance 

Processing of global assay parameters using ISURF software #03407 (PREXCEL-Q) 

Consultation on detecting and avoiding RT and PCR inhibition for all sample types and isolation methods 

File system creation and initial qPCR Test Plate set-up printouts and consultation 

Processing of Test Plate results into final set-up parameters and procedural printouts for final sample qPCR using ISURF software #03407 (PREXCEL-Q)  

Excel spreadsheets custom-created for EAMP-corrected data analysis and graphing 

All plant and animal species qPCR considered   

Service by appointment

Brugia malayi is a roundworm nematode, one of the three causative agents of lymphatic filariasis in humans. Lymphatic filariasis, also known as elephantiasis, is a condition characterized by swelling of the lower limbs. The two other filarial causes of lymphatic filariasis are Wuchereria bancrofti and Brugia timori, which differ from B. malayi morphologically, symptomatically, and in geographical extent.[1]

B. malayi is transmitted by mosquitoes and is restricted to South and South East Asia. It is one of the tropical diseases targeted for elimination by the year 2020 by the World Health Organization, which has spurred vaccine and drug development, as well as new methods of vector control.

Brugia Test Plate: 3 targets

BrugiaKimber-Song

Additional examples (out of >100 examples since 2001)

Book Chapter: “Difficult Templates and Inhibitors of PCR”Running Title: qPCR inhibition and amplification of difficult templates May 2010

Table of ContentsIntroduction

Clarification of termsImportance of eliminating inhibition from qPCR assays

Cq-based standard curve method vs. the sigmoidal curve-fitting method and Cyo methodThe quasi-exponential nature of kinetic fluorogenic qPCR efficiency estimates

Causes of InhibitionThe “sample effect”

InhibitorsRT, PCR and qPCR inhibitors in soil and plant material

RT, PCR and qPCR inhibitors in animal materialOperator-introduced variation

Sample isolation and RT and/or qPCR-inhibitory material The “inhibitory characteristic” of a sample

Inhibition issues specific to RNA and RT reactionsThe fidelity of reverse transcription (RT) reactions

RT-PCR reaction set up and optimizationDifficult TemplatesGC-rich templatesAT-rich templatesRepetitive DNA

Additives and enhancers for PCR, RT-PCR and qPCRLists of additives are next in bold heading

PREXCEL-QP-Q proof of principle in minimizing the “operator effect”

The 3’:5’ and SPUD AssaysqPCR and Music

AppendixReferences

Acknowledgements:

Dr. Mark R. Ackermann and 80+ other researchers whose qPCR projects

inspired the creation of the PQ program