Application Note: Real-time PCR Study Report on Nancy-520

Product No. 01494

Background

Sigma-Aldrich offers a fluorescent stain for dsDNA called Nancy-520, a greener alternative to Ethidium bromide. This dye has also been successful in PCR (Real time-PCR) applications. In order to develop an optimal qPCR (quantitative PCR) master mix with Nancy-520, for labeling of PCR products, two studies were conducted by an external research institute, IBR Inc. (Institute for Biopharmaceutical Research), at the request of Sigma-Aldrich.

Experimental Design

Study 1: This study compared the sensitivity and efficiency of Nancy-520 with the standard intercalating dye SYBR® Green I in a qPCR. The use of qPCR allows the detection of the amplicon in real time. The fluorescent dye (SYBR Green I or Nancy-520) binds to double-stranded DNA (PCR product) and emits light upon excitation. As a PCR product accumulates, more dye binds to the product, resulting in an increase in the emission. Based on its fluorescent properties (excitation λ = 520 nm and emission λ = 560 nm), IBR Inc. evaluated the suitability of Nancy-520 as an alternative to SYBR Green I in qPCR.

In this study, PBMC (Peripheral Blood Mononuclear Cells) were isolated from 5 mL of blood using Ficoll® gradient centrifugation. The DNA eluate from PBMC ranging from 1.5 ×104 to 9.13 × 10–1 cell equivalents was tested with two sets of primers. One primer set amplified the multicopy sequence Alu repeat and another primer set amplified the single copy gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). The PCR reaction mix was prepared with Jumpstart™ Taq ReadyMix™ (Product No. P2893) or SYBR® Green JumpStart™ Taq ReadyMix™ (Product No. S4438) from Sigma Aldrich. The final concentration of Nancy-520 (Product No. 01494) and SYBR® Green I (Product No. S9430) was 1× per each PCR reaction when using the Jumpstart™ ReadyMix™ Taq. The qPCR reaction was performed in a 7500 Real Time PCR System (TaqMan® 7500, Applied Biosystems™, Type AB7500). For this experiment, three filters were chosen after a filter suitability test was conducted with the filters available with the TaqMan® 7500, Applied Biosystems™, Type AB7500 instrument. The three filters that were found to be suitable for the detection of DNA amplicons stained with Nancy-520 were Cy3®, JOE™, and VIC®. For the detection of DNA stained with SYBR® Green I, the SYBR® Green I filter was used.

Study 2: A second study was conducted to develop an optimized Nancy-520 qPCR master mix for direct DNA amplification, detection, and quantification without the use of gel electrophoresis. The DNA was isolated from PBMC and was tested with two primer sets, one set that amplifies the Alu repeat and another primer set that amplifies GAPDH gene, similar to study 1. For the determination of the optimal concentration of Nancy-520, five master mixes were prepared with Jumpstart™ ReadyMix™ Taq, varying in the concentration of Nancy-520 (0.5× to 5×). The qPCR was performed with the TaqMan® 7500, Applied Biosystems, Type AB7500 using the VIC® filter.

For optimization of the protocol, 10 mL of a master mix supplemented with 2.5x Nancy-520 was used to determine reproducibility in terms of inter-assay variance (day-to-day and person-to-person). These experiments were conducted after the determination of the range of primer concentration to be used with the master mix by performing a primer titration experiment (0.05–2 µM primer set for Alu or GAPDH).

For the reproducibility experiments using the Nancy-520 master mix, day-to-day and operator-to-operator experiments were conducted with the final primer concentration of 1 µM for GAPDH and 2 µM for Alu sequence based on the primer titration experiment.

For the day-to-day experiment, one operator prepared an identical qPCR experiment on two different days. To determine operator-to-operator variance, two operators prepared an identical qPCR experiment. Both the master mixes contained five concentrations of the DNA template. The data evaluation was performed with the 7500 system SDS software.

Results

Study 1: The results from using Nancy-520 and SYBR® Green I in qPCR have indicated that Nancy-520 is more efficient and sensitive in DNA detection with the filters Cy3®, JOE™, and VIC ® (for Nancy-520) and the SYBR® Green I filter (for SYBR Green I), and with the primer set for GAPDH. The efficiency of the qPCR with the primer set for the Alu sequence was observed to be below the acceptance criterion. This may be as a result of fast depletion of the primers due to the high abundance of the sequence/genome. The use of Nancy-520 has also been found to be more efficient when compared to the use of commercially available ready-to-use SYBR® Green JumpStart™ Taq ReadyMix™.

Study 2: The efficiency of the qPCR with GAPDH primer set was more when compared to the amplification of the Alu sequence. This may be due to the generation of primer dimers with the use of primer set for multicopy sequence Alu repeat, which get amplified by DNA polymerase, leading to the generation of non-specific DNA. The best result was observed when the final concentration of Nancy-520 was 2.5× in the qPCR mix with the primer set for GAPDH gene. This concentration was further used for the primer titration assay and to determine inter-assay variance.

In the primer titration assay with varying primer concentration (0.05–2 µM), the primer set for GADPH gene produced efficient results at 0.1–1 µM concentration and the primer set for Alu sequence showed efficiency at a higher concentration of 2 µM.

It was also observed during the inter-assay variance experiments that the results from day 1 and day 2 by operator 2 could be reproduced. The results from operator 1 could also be reproduced by operator 2. These results indicate that 2.5× master mix produces a set of robust data.

Conclusion

From the experiments conducted by IBR Inc., it can be concluded that Nancy-520 is more efficient and sensitive in DNA detection when compared to SYBR® Green I in qPCR. A 2.5× Nancy-520 master mix produces reliable, efficient and reproducible results with the primer set for GADPH gene (0.1–1 µM) and the primer set for Alu sequence (2 µM) when detected with the VIC® filter.

SYBR is a registered trademark of Life Technologies Corporation.
Ficoll is a registered trademark of GE Healthcare
JumpStart is a trademark of Sigma-Aldrich Co. LLC
ReadyMix is a trademark of Sigma-Aldrich Co. LLC
TaqMan is a registered trademark of Roche Molecular Systems, Inc
Applied Biosystems is a registered trademark of Life Technologies, Inc.
Cy3 is a registered trademark of GE Healthcare
VIC and JOE are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries

Date Published: October, 19, 2015
Product Manager
: Monika Baeumle
Author
: Yashaswini J V