Application Note

Sensitive fluorescent quantitation of DNA with the Quant-iT PicoGreen dsDNA Assay Kit

  • Sensitive fluorescent quantitation of DNA down to 50 pg/mL
  • Linear dynamic range spanning over four orders of magnitude
  • Easy analysis of results with preconfigured protocol in SoftMax Pro Software

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Joyce Itatani | Application Scientist | Molecular Devices
Cathy Olsen | Sr. Application Scientist | Molecular Devices

Introduction

Double-stranded DNA is typically quantitated in microplate readers by measuring the absorbance of the DNA solution at 260 nm. However, this method is only able to measure down to about 250 ng/mL on a typical absorbance microplate reader. For biological applications involving small samples, such as next-generation sequencing and quantitation of DNA amplification products, more sensitive methods are needed. The Quant-iT PicoGreen dsDNA Assay Kit from Thermo Fisher Scientific is more specific for DNA and is about 1000 times more sensitive than traditional absorbance methods. The dynamic range of this assay in microplate format, as stated in the product manual, is from 250 pg/mL to 1000 ng/mL with a single dye concentration. Here, we demonstrate that with Molecular Devices SpectraMax® microplate readers and the Quant-iT PicoGreen assay, users can reliably measure concentrations as low as 50 pg/mL of double-stranded DNA.

To maximize sensitivity of the assay, it is necessary to use optimal excitation and emission wavelengths. Unlike filter-based plate readers, the dual monochromators in the SpectraMax iD5 Multi-Mode Microplate Reader and other SpectraMax readers allow the selection of any wavelength within the reader’s stated range. It is important to determine the excitation and emission wavelengths that provide the best sensitivity and dynamic range for the assay, since these may differ somewhat from those used with filter-based plate readers, and from wavelengths recommended in the kit manual.

Materials

Methods

Instrument and protocol setup

Prepare the assay

The method for this assay follows the instructions in the product information sheet for QuantiT PicoGreen dsDNA Reagent and Kits, except the assay volume is proportionately reduced from 2.0 mL to 200 μL to fit a 96-well microplate format.

Note: In some cases, it may be preferable to make the standard curve using DNA similar to the type being assayed.

Parameter
SpectraMax iD5/iD3
SpectraMax i3x
SpectraMax M5/M5e/M4/M3/M2/M2e
SpectraMax Gemini EM/XPS
SpectraMax Mini
Read mode
Fluorescence (FL)
Fluorescence (FL)
Fluorescence (FL)
Fluorescence (FL)
Fluorescence (FL)
Read type
Endpoint
Endpoint
Endpoint
Endpoint
Endpoint
Wavelengths
Excitation: 485 nm
Emission: 535 nm
Excitation: 490 nm,
bandwidth 9 nm
Emission: 525 nm,
bandwidth 15 nm
Excitation: 490 nm,
Emission: 525 nm,
Emission cutoff: 515 nm
Excitation: 490 nm,
Emission: 525 nm,
Emission cutoff: 515 nm
Fluorescence filter cube: FL-535 (Excitation 485 nm, Emission 535 nm, Dichroic 508 nm)
Plate type and Read area
Select based on microplate and wells used
Select based on microplate and wells used
Select based on microplate and wells used
Select based on microplate and wells used
Select based on microplate and wells used
PMT and Optics
PMT gain: Automatic
Integration time: 200 ms
Read height: Optimize for microplate used
PMT gain: N/A
Flashes per read: 10
Read height: Optimize for microplate used
PMT gain: Automatic
Flashes per read: 10
PMT gain: Automatic
Flashes per read: 10
PMT gain: Automatic
Integration time: 400 ms
Read height: Optimize for microplate used

Table 1. Instrument settings for SpectraMax readers. For SpectraMax iD5, i3x, and Mini readers, the read height setting should be optimized for the microplate used. Note: Additional readers with similar performance are listed.

Read the microplate

Analyze the data

Sample
DNA conc (ng/mL)
Average RFU
StdDev
%CV
1
1000.00
54893498
611149.3
1.1
2
333.333
17114392
685401.0
4.0
3
111.111
5884808
249140.5
4.2
4
37.037
1938706
21246.0
1.1
5
12.346
667849
10336.2
1.5
6
4.115
231812
9389.3
4.1
7
1.372
74577
4324.4
5.8
8
0.457
25963
2403.5
9.3
9
0.152
8981
1422.3
15.8
10
0.051
2441
1205.6
49.4

Table 2. DNA standards.

Results

DNA standards ranging from 50 pg/mL to 1 μg/mL were detected using the Quant-iT PicoGreen dsDNA Assay Kit and SpectraMax readers (data from the SpectraMax iD5 reader are shown, but other SpectraMax readers gave similar results). SoftMax Pro Software automatically calculated average RFU, standard deviation, and %CV for each set of standard replicates. A standard curve was plotted using the log-log curve fit in SoftMax Pro Software (Figure 1). Sensitivity down to 50 pg/mL was observed using the 96-well microplate format and standard limit of detection calculation of three times standard deviation of the blank. This is well below the lower limit of 250 pg/ mL stated in the Quant-iT PicoGreen assay product insert. Figure 2 shows the high-range (A) and low-range (B) standard curves. Linearity was excellent throughout the standards’ range (r2 ≥ 0.99 for each curve shown).

DNA standard curve

Figure 1. DNA standard curve. DNA standards ranging from 50 pg/mL to 1000 ng/mL were assayed using the Quant-iT PicoGreen dsDNA Assay Kit on the SpectraMax iD5 reader. The standard curve was plotted using the log-log curve fit in SoftMax Pro Software (r2 = 1.00).

High-range and low-range standard curves

Figure 2. High-range (A) and low-range (B) standard curves. Curves were plotted using the log-log curve fit in SoftMax Pro Software (both curves, r2 = 0.99).

Conclusion

The Quant-iT PicoGreen dsDNA Assay Kit, when run on a SpectraMax microplate reader with SoftMax Pro Software, is a quick, sensitive detection method for double-stranded DNA. The analysis capabilities of the software provide quantitation in an easy-to-read, user customizable report format. A preconfigured protocol is available in the software to facilitate rapid assay setup.

Joyce Itatani | Application Scientist | Molecular Devices
Cathy Olsen | Sr. Application Scientist | Molecular Devices

简介

双链 DNA 通常通过测定 DNA 溶液在 260 nm 处的吸光度在 酶标仪中进行定量。然而,该方法在典型的光吸收酶标仪上只 能测量到约 250 ng/mL。对于涉及小样本的生物应用,如下 一代测序和 DNA 扩增产物的定量,需要更敏感的方法。来自 Thermo Fisher Scientific 公司的 Quant-iT PicoGreen dsDNA 检测试剂盒对 DNA 更特异,比传统光吸收方法的灵敏度高约 1000 倍。 如产品手册所述,此实验在微孔板格式中采用单 一染料浓度的动态范围从 250 pg/mL 到 1000 ng/mL。在这 里,我们证明了使 用 Molecular Devices 公司 SpectraMax® 酶标仪和 Quant-iT PicoGreen 实验,用户可以可靠地测量低 至 50 pg/mL 的双链 DNA 浓度。

为了最大化实验的灵敏度,需要使用最佳的激发和发射波长。 与基于滤光片的酶标仪不同,SpectraMax iD5 多功能酶标仪 和其他 SpectraMax 酶标仪中的双单色器允许在酶标仪规定 的范围内选择任何波长。确定为实验提供最佳灵敏度和动态 范围的激发和发射波长是很重要的,因为这些波长可能与基 于滤光片的酶标仪使用的波长以及试剂盒手册中推荐的波长 有所不同。

材料

方法

仪器和方案设置

实验准备

该方法遵循 QuantiT PicoGreen dsDNA 试剂和试剂盒产品信 息表的说明,除了实验体积从 2.0 mL 按比例减少到 200µL, 以适应 96 孔微孔板格式。

注 : 在某些情况下,最好使用与被测类型相似的 DNA 做标准 曲线。

Parameter
SpectraMax iD5/iD3
SpectraMax i3x
SpectraMax M5/M5e/M4/M3/M2/M2e
SpectraMax Gemini EM/XPS
Read mode
Fluorescence (FL)
Fluorescence (FL)
Fluorescence (FL)
Fluorescence (FL)
Read type
Endpoint
Endpoint
Endpoint
Endpoint
Wavelengths
Excitation: 485 nm
Emission: 535 nm
Excitation: 490 nm,
bandwidth 9 nm
Emission: 525 nm,
bandwidth 15 nm
Excitation: 490 nm,
Emission: 525 nm,
Emission cutoff: 515 nm
Excitation: 490 nm,
Emission: 525 nm,
Emission cutoff: 515 nm
Plate type and Read area
Select based on microplate and wells used
Select based on microplate and wells used
Select based on microplate and wells used
Select based on microplate and wells used
PMT and Optics
PMT gain: Automatic
Integration time: 200 ms
Read height: Optimize for microplate used
PMT gain: N/A
Flashes per read: 10
Read height: Optimize for microplate used
PMT gain: Automatic
Flashes per read: 10
PMT gain: Automatic
Flashes per read: 10

***表 1 SpectraMax 酶标仪的仪器设置。*对于 SpectraMax iD5 和 i3x 酶标仪,读取高度设置应针对所使用的微孔板进行优化。注 : 列出了其他具有类似 性能的酶标仪。

读板

分析数据

Sample
DNA conc (ng/mL)
Average RFU
StdDev
%CV
1
1000.00
54893498
611149.3
1.1
2
333.333
17114392
685401.0
4.0
3
111.111
5884808
249140.5
4.2
4
37.037
1938706
21246.0
1.1
5
12.346
667849
10336.2
1.5
6
4.115
231812
9389.3
4.1
7
1.372
74577
4324.4
5.8
8
0.457
25963
2403.5
9.3
9
0.152
8981
1422.3
15.8
10
0.051
2441
1205.6
49.4

表 2 DNA 标准品。

结果

使用 Quant-iT PicoGreen dsDNA 检测试剂盒和 SpectraMax 酶 标 仪 检 测 50 pg/mL 至 1 µg/mL 的 DNA 标 准 品 ( 数 据 来 自 SpectraMax iD5 酶 标 仪,但 其他 SpectraMax 酶 标 仪 给 出类似的结果 )。SoftMax Pro 软件自动计算每组标准品重复 的平均 RFU,标准偏差和 %CV。在 SoftMax Pro 软件中使用 对数 - 对数曲线拟合绘制了一条标准曲线 ( 图 1 )。采用 96 孔 微孔板格式,检测限为空白样品标准偏差的 3 倍,灵敏度可低 至 50 pg/mL。这远远低于 Quant-iT PicoGreen 检测产品说 明书中规定的 250 pg/ mL 的下限。图 2 为高范围 (A) 和低范 围 (B) 的标准曲线。在标准品范围内线性良好 ( 图中各曲线的 r 2 ≥ 0.99 )。

DNA standard curve

图 1 DNA 标准曲线。 采用 Quant-iT PicoGreen dsDNA 检测试剂盒, 在 SpectraMax iD5 酶 标 仪 上 检 测 50 pg/mL 至 1000 ng/mL 范 围 内 的 DNA 标准品。采用 SoftMax Pro 软件中的对数 - 对数曲线拟合 (r2=1.00) 绘制标准曲线。

High-range and low-range standard curves

***图 2 高范围 (A) 和低范围 (B) 标准曲线。*使用 SoftMax Pro 软件中的对数 - 对数曲线拟合绘制曲线 ( 两条曲线均为 r2=0.99 )。

总结

Quant-iT PicoGreen dsDNA 检测试剂盒在 SoftMax Pro 软件的 SpectraMax 酶标仪上运行时,是一种快速、灵敏的双链 DNA 检测方法。该软件的分析功能以易于阅读、用户可自定义的报告格式提供定量。一个预先配置的方案在软件中可用,以方便快速 检测设置。

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