Nucleic Acid Extraction Control in Real-Time PCR Assays

1. Nucleic Acid Extraction Control in Real-Time PCR Assays • Steve Hawkins • Senior Global Product Manager • Bioline

2. Real-Time PCR in Diagnostics Advantages Challenges False Positive Results Contamination • Speed (hours vs. days) • Sensitivity • Specificity • Throughput • Multiplexing • False Negative Results • – PCR Inhibition • – Nucleic Acid Extraction • Failure

3. DNA Extraction Control (DEC)

4. Process of incorporating DECinto real-time PCR Assay

5. Inefficient DNA extraction Inefficient DNA extraction was simulated by substitution of either the lysis buffer or binding buffer with PBS. Reference gene (green channel) / Internal control (red channel) SensiFAST SYBR Complete lysis step (red), no lysis (green) and no binding buffer (orange) showed significant “loss of sample” This demonstrate that the DEC can be used to monitor DNA extraction.

6. DEC vs. pure DNA DEC and DNA were spiked into cell resuspension. Extraction was carried out with or without lysis buffer in parallel for simulation of inefficient extraction. Gene of interest (green channel) / Internal control (red channel) SensiFAST SYBR Sample with (red) or without a lysis step (violet), a difference, but there was no change of Ct in the naked DNA, with (green) or without a lysis step (blue) Spike pure DNA into sample as internal control may not function as extraction control due to the lack of lysis required.

7. PCR reaction inhibition EDTA was added into elution buffer, as an inhibitory agent to show the effects on the internal control. Gene of Interest Internal DEC control SensiFAST SYBR DEC shows consistent inhibition level as with the sample target. Inhibition of PCR reaction can be identified using DEC.

8. Minimal interference and consistency A triplex reaction was compared with singleplex reactions to look for interference by the DEC. SensiFAST SYBR The results illustrate the consistency of the DEC

9. RNA Extraction Control (REC)

10. REC, spiked DNA and extraction control Spiked DNA control and REC were co-extracted out +/- lysis buffer or +/- EtOH (critical step in RNA Kit prep.). No DNase digestion was performed Spiked DNA Spiked DNA control will always produce a positive signal, so therefore no value as an extraction control REC Only REC demonstrates the effect of extraction quality on the Ct and is an indicator of RT inhibition SensiFAST SYBR One-Step

11. REC monitors PCR inhibition Different concentrations of EDTA were added prior to elution, as an inhibitory agent to test the monitoring capability of the internal control. SensiFAST SYBR One-Step The presence of PCR inhibitors result in a shift in Ct

12. REC provides reproducible results Three REC samples were extracted in parallel. The multiple extractions were performed and analyzed by real-time SensiFAST SYBR One-Step The results illustrate the reproducibility over several fold dilutions of template and over a number of extractions

13. Summary • Nucleic acid extraction process monitoring is important in reducing false negative results • Pure DNA controls not effective in monitoring nucleic acid extraction process • DEC and REC effective in monitoring nucleic acid extraction and PCR inhibition

14. Summary • Monitors extraction efficiency • Monitors PCR inhibition • Consistent signal in a validated assay • Easy incorporation steps • Minimize exogenous contaminant introduction

15. AcknowledgementsDr. Lopeti LavuloTom LinLisa YangDr. Sally Dubedat (Royal Prince Alfred Hospital, Sydney)