PREXCEL-Q proof of principle and the formation of the ISU qPCR Consultation Service

1. PREXCEL-Q proof of principle and the formation of the ISU qPCR Consultation Service Jack M.Gallup

2. PREXCEL-Q precepts: • Use “Stock I” (sample mixture) on a “Test Plate” to: 1.) Determine the ‘inhibitory threshold’ (minimum dilution required by samples to avoid qPCR inhibition). 2.) Identify the valid dilution range(s) of “Stock I” for each target standard curve. 3.) Determine the “Tier 1” dilution for each sample, individually; per target (if necessary): 4.) Determine if more than 1 “Tier dilution” is needed to accommodate rare or hyper-abundant targets.

3. “Tier 1” (the red-dot)

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

6. LOG-linear ranges identified for each target

7. Best case scenario: For simplicity’s sake, try to find the same “Homodynamic” range for all targets

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

9. 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 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. What is it? What does it do? How does it work?Is it easy to use? Is it free? …Yes! … • 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)

10. 11 serial dilutions: from full-strength Stock I to 1:50,000 or 100,000 Test Plate N T C W E L L S N T C W E L L S P-Q Cq analysis Final Plates: df = (Upper/Lower)1/(p - 1) Stock I used for standards Unknowns diluted to respective Tier ng/uL spots

11. qPCR Consultation Service http://vetmed.iastate.edu/isuqpcrconsultationservices

12. ISU qPCR Consultation Service • For qPCR Assay Development and Project ManagementIn 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

13. Brugia Test Plate: 3 targets Additional examples (out of >100 examples since 2001) Brugia malayi is a roundwormnematode, 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 Kimber-Song

24. Book Chapter: “Difficult Templates and Inhibitors of PCR” Running Title: qPCR inhibition and amplification of difficult templates May 2010 Table of Contents Introduction Clarification of terms Importance of eliminating inhibition from qPCR assays Cq-based standard curve method vs. the sigmoidal curve-fitting method and Cyo method The quasi-exponential nature of kinetic fluorogenic qPCR efficiency estimates Causes of Inhibition The “sample effect” Inhibitors RT, PCR and qPCR inhibitors in soil and plant material RT, PCR and qPCR inhibitors in animal material Operator-introduced variation Sample isolation and RT and/or qPCR-inhibitory material The “inhibitory characteristic” of a sample Inhibition issues specific to RNA and RT reactions The fidelity of reverse transcription (RT) reactions RT-PCR reaction set up and optimization Difficult Templates GC-rich templates AT-rich templates Repetitive DNA Additives and enhancers for PCR, RT-PCR and qPCR Lists of additives are next in bold heading PREXCEL-Q P-Q proof of principle in minimizing the “operator effect” The 3’:5’ and SPUD Assays qPCR and Music Appendix References Acknowledgements: Dr. Mark R. Ackermann and 80+ other researchers whose qPCR projects inspired the creation of the PQ program

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