Miniaturizing Real-Time PCR with the Nanodrop™

Cost Reduction with Conservation of Sample

PCR has become a widely used methodology for the analysis of gene expression and genotyping. Key factors that influence use of this technique include high reagent costs and the availability of limited sample.

To address these concerns, the performance of the Nanodrop™ dispenser from Innovadyne Technologies, Inc. was evaluated using a TaqMan® assay in a 384-well format. To demonstrate the application of non-contact, nanoliter dispensing to real-time PCR, amplification of the single copy RNase P gene from human genomic DNA, a common verification reference, was chosen as the target for the assay.

384 replicate real-time PCR reactions were set up using traditional liquid handling robots in a 10µL total volume while the Nanodrop was used to assemble 1.25µL total volume reactions using the same components.

TaqMan® Assay Assembly

Dispenser

Conventional

Nanodrop™

Real Time Cycler

ABI Prism™ 7900HT with 384 Well Plate Module

TaqMan® Universal Master Mix, No AmpErase®UNG

5 µL

625 nL

Nuclease Free Water

1 µL

125 nL

5X Primer-Probe Mix*

2 µL

250 nL

Human Genomic DNA at 0.5ng/uL

2 µL

250 nL

Total Volume

10 µL

1.25 µL

Reactions were cycled using identical 40 cycle amplification profiles in ABI Prism™ 384-Well Clear Optical Reaction Plates.

* Probe consisted of a 5' FAM™ reporter with a 3' TAMRA™ quencher.

Analysis of the data shows that the Nanodrop delivers comparable results while significantly reducing reagent costs and consumption of sample.

Cycle Threshold (Ct) Values for 384 Replicates

10µL Conventional Dispensing

1.25µL Nanodrop Dispensing

Cycle Threshold (Ct) Metric
*Total Volume*	*10 µL*	*1.25 µL*
Ct Mean	28.82	29.03
Ct St.Dev	0.22	0.30
Ct %CV	0.77	1.02
Ct Minimum	28.09	28.13
Ct Maximum	29.29	29.99
Ct Range	1.20	1.86

Reagent Cost Savings

Reagent Cost Savings
	*10 µL Assay*	*1.25 µL Assay*
Cost per well	$0.54	$0.072
Cost per plate	$207	$26
Savings/plate	$181
Savings/500 plates*	$90,500
* Potential savings

Summary of Advantages

Minimizes consumption of samples
Drastically reduces reagent costs
Produces robust, low-volume assays
Supports the 96-, 384- and 1536-well formats
Flexible assay designs

Avoiding Carryover During PCR Assembly

Prevention of sample cross contamination is a major concern for laboratories utilizing liquid handling robotics to set up PCR reactions. To address the issue when using the Nanodrop Dispenser, several tip wash routines were evaluated for the capacity to eliminate sample carryover.

Briefly, 2.5 µL TaqMan® assays for the human RNase P gene were assembled using the Nanodrop. DNA samples and corresponding blank controls were dispensed alternately across 8 consecutive columns of a 384-well plate. One of four wash routines was performed subsequent to each DNA addition. Wash effectiveness was then evaluated by assessing real-time amplification Cycle Threshold (Ct) Values in the corresponding blank control wells.

The data show that the Nanodrop is capable of performing PCR assembly without carryover. A wash with 2.5 mL of water alone is sufficient to prevent carryover contamination while additional treatments combining a 2.5 mL water wash with either a 2% Micro90 detergent or a 2% bleach wash is also effective, and importantly, does not inhibit reaction performance.

Experimental Procedure

Step 1: Dispense 1.25 µL of Taqman® Universal Master Mix (No AmpErase® UNG) to all wells
Step 2: Dispense 250 nL of Nuclease Free Water to all wells
Step 3: Dispense 500 nL of 5X Primer-Probe Mix to all wells
Step 4: Dispense 500 nL of DNA sample at 0.5 ng/µL to alternating wells of a single column
Step 5: Perform specified wash treatment
Step 6: Dispense water blank to corresponding wells of the adjacent column
Step 7: Repeat steps 4-6 for all wash treatments
Step 8: Cycle and read reactions in an ABI PrismTM 7900HT with a 384-Well Module using a 40 cycle amplification profile

TaqMan® Reaction Assembly
*Component*	*Addition*	*Volume*
TaqMan® Universal Master Mix, No AmpErase®UNG	1st	1.25 µL
Nuclease Free Water	2nd	250 nL
5X Primer-Probe Mix*	3rd	500 nL
DNA Template or Water Blank	4th	500 nL
Total Volume		2.5 µL
* Probe consisted of a 5’ FAM™ reporter with a 3’ TAMRA™ quencher

Wash Treatments
Wash I	Wash with 0.6 mL/tip of water
Wash II	Wash with 2.5 mL/tip of water
Wash III	Wash with 0.5 mL/tip of 2% Micro 90 detergent followed by 2.5 mL/tip of water
Wash IV	Wash with 0.5 mL/tip of 2% bleach followed by 2.5 mL/tip of water

Cycle Threshold Values for DNA Samples and Blank Controls

Summary of Advantages

Assembly of PCR based assays without carryover
Simple wash routines
Eliminates disposable tip costs
Reduces plastic waste in the environment

Home Applications PCR/Sequencing Miniaturizing RT-PCR with the Nanodrop

TaqMan® Assay Assembly
*Dispenser*	*Conventional*	*Nanodrop™*
Real Time Cycler	ABI Prism™ 7900HT with 384 Well Plate Module
TaqMan® Universal Master Mix, No AmpErase®UNG	5 µL	625 nL
Nuclease Free Water	1 µL	125 nL
5X Primer-Probe Mix*	2 µL	250 nL
Human Genomic DNA at 0.5ng/uL	2 µL	250 nL
Total Volume	10 µL	1.25 µL
Reactions were cycled using identical 40 cycle amplification profiles in ABI Prism™ 384-Well Clear Optical Reaction Plates. * Probe consisted of a 5' FAM™ reporter with a 3' TAMRA™ quencher.