PicoGreen是一种特异性结合双链DNA (dsDNA) 的饱和荧光染料，激发波长为480 nm，发射波长为520 nm，与大多数荧光计和荧光酶标仪兼容。 PicoGreen仅与dsDNA结合后才发出荧光， 不受核苷酸和单链核酸的影响， 产生的荧光强度与dsDNA浓度成正比， 且无序列依赖性， 非常适合于dsDNA的定量检测， 可检出25 pg/ml~1 μg/ml浓度范围内的dsDNA。
由于PicoGreen灵敏度高、特异性强、耐受性好、无需纯化DNA，从2010版中国药典开始，已被列为生物制品DNA残留检测的标准方法之一。 根据药典说明，当dsDNA浓度在1.25 ng/ml~80 ng/ml范围内时线性最好 (R2 >0.99)。
使用方法
1. 染料工作液配制
PicoGreen原液以浓缩液形式保存在无水的DMSO (二甲基亚砜)中。实验时，一般应将原液用1× TE按1:100 的比例稀释，配制成2× 染料工作液，然后按1: 1比例与待测样品混合，使PicoGreen终浓度为1 ×。由于本试剂容易吸附到玻璃表面，应在塑料容器中配制，并注意尽量避光。染料工作液最好在配制好数小时内使用，以保证最佳结果。
2 ．实验方法
① 标准液配制：
取小牛胸腺DNA干粉1 mg (Tris, NaCl等浓度已成标准体系)，加入1 ml双蒸水，配制成1 mg/ml的标准液。
② 染料工作液配制：
取10 μl PicoGreen原液，加入990 μl 1× TE缓冲液，配制成2×染料工作液。
③ 标准品工作液配制：
a. 母液稀释：取10 μl (1 mg/ml)标准液加入到990 μl 1× TE缓冲液中， 稀释成10 μg/ml标准品工作液； 再取10 μl (10 μg/ml)标准品工作液液加入到990 μl 1× TE缓冲液中，稀释成100 ng/ml的标准品工作液。
b. 倍比稀释：取800 μl (100 ng/ml)的标准品工作液加入到200 μl 1× TE缓冲液中，浓度为80 ng/ml；再取 500 μl (80 ng/ml)标准品工作液加入到500 μl 1× TE缓冲液中，稀释到40 ng/ml；依次倍比稀释，配成20 ng/ml、10 ng/ml、5.0 ng/ml、2.5 ng/ml、1.25 ng/ml的标准品工作液。
④ 标准曲线绘制：
分别将倍比稀释后的1.25 ng/ml~80 ng/ml标准品工作液和2 ×染料工作液按1:1体积混匀 (具体体积根据荧光设备和容器的要求确定)，避光、室温放置5 min。使用荧光仪检测样品的荧光值：将混合后的溶液加入微量比色皿，注意不要在样品中引入气泡，轻弹比色皿外部以驱散气泡。以1× TE缓冲液为空白对照，设定激发波长480 nm，发射波长520 nm，在恒定温度下测定样品和空白对照的荧光值。或者使用96孔酶标板在荧光酶标仪中检测。检测结束后，以各浓度荧光值减去空白对照荧光值，再与标准品溶液浓度 (ng/ml)进行线性回归，绘制标准曲线。
⑤ 待测样品浓度测定：
将待测样品溶液和2×染料工作液按1:1体积混匀， 采用④中同样的方法，在相同温度下测定待测样品的荧光强度 (必要时先使用TE缓冲液将待测样品稀释到PicoGreen线性范围之内)，根据标准曲线的线性回归方程，计算待测样品浓度。
注意事项
1. 荧光染料均存在淬灭问题， 请尽量注意避光，以减缓荧光淬灭；
2. PicoGreen工作液最好现配现用，以保证最佳结果；
3. PicoGreen荧光强度受温度影响较大，应保证绘制标准曲线和样品测定在同一恒定温度下进行；
4. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
PicoGreen is a saturating fluorescent dye that specifically binds to double-stranded DNA (dsDNA). It has an excitation wavelength of 480 nm and an emission wavelength of 520 nm, and is compatible with most fluorometers and fluorescent microplate readers. PicoGreen only fluoresces upon binding to dsDNA, and is not affected by nucleotides or single-stranded nucleic acids. The resulting fluorescence intensity is proportional to the dsDNA concentration in a sequence-independent manner, making it highly suitable for quantitative detection of dsDNA. It can detect dsDNA in the concentration range of 25 pg/ml to 1 μg/ml.
Due to its high sensitivity, strong specificity, good tolerance, and no requirement for DNA purification, PicoGreen has been listed as one of the standard methods for residual DNA detection in biological products since the 2010 edition of the Chinese Pharmacopoeia. According to the pharmacopoeia, the linearity is optimal (R² > 0.99) when the dsDNA concentration ranges from 1.25 ng/ml to 80 ng/ml.
Protocol
1. Preparation of Dye Working Solution
PicoGreen stock solution is supplied as a concentrate in anhydrous DMSO (Dimethyl Sulfoxide). For experiments, the stock solution is generally diluted 1:100 with 1× TE buffer to prepare a 2× dye working solution, which is then mixed with samples at a 1:1 ratio to achieve a final 1× PicoGreen concentration. As this reagent readily adsorbs to glass surfaces, preparation should be performed in plastic containers with protection from light as much as possible. The dye working solution is best used within several hours of preparation for optimal results.
2. Experimental Procedure
① Preparation of Standard Solution
Weigh 1 mg of Sigma calf thymus DNA powder (with standardized concentrations of Tris, NaCl, etc.), add 1 ml of double-distilled water to prepare a 1 mg/ml standard solution.
② Preparation of Dye Working Solution
Take 10 μl of PicoGreen stock solution and add 990 μl of 1× TE buffer to prepare a 2× dye working solution.
③ Preparation of Standard Working Solutions
a. Stock dilution: Take 10 μl of 1 mg/ml standard solution and add to 990 μl of 1× TE buffer to dilute to a 10 μg/ml standard working solution. Then take 10 μl of the 10 μg/ml standard working solution and add to 990 μl of 1× TE buffer to dilute to a 100 ng/ml standard working solution.b. Serial dilution: Take 800 μl of 100 ng/ml standard working solution and add to 200 μl of 1× TE buffer to obtain 80 ng/ml. Then take 500 μl of 80 ng/ml standard working solution and add to 500 μl of 1× TE buffer to dilute to 40 ng/ml. Continue serial dilutions to prepare standard working solutions of 20 ng/ml, 10 ng/ml, 5.0 ng/ml, 2.5 ng/ml, and 1.25 ng/ml.
④ Standard Curve Construction
Mix the serially diluted standard working solutions (1.25 ng/ml to 80 ng/ml) with the 2× dye working solution at a 1:1 volume ratio (adjust volume according to fluorometer and container requirements). Incubate at room temperature for 5 min protected from light. Measure the fluorescence using a fluorometer: add the mixed solution to a microcuvette, avoid introducing air bubbles, and gently tap the outside of the cuvette to remove bubbles. Use 1× TE buffer as blank control, set excitation wavelength to 480 nm and emission wavelength to 520 nm, and measure fluorescence of samples and blank at a constant temperature. Alternatively, detection can be performed in a 96‑well microplate using a fluorescent microplate reader. After detection, subtract blank fluorescence from each sample reading, perform linear regression against standard solution concentrations (ng/ml), and construct a standard curve.
⑤ Determination of Sample Concentration
Mix the test sample solution with the 2× dye working solution at a 1:1 volume ratio. Measure the fluorescence intensity under the same temperature and conditions as in step ④ (dilute samples with TE buffer into the linear range of PicoGreen if necessary). Calculate the sample concentration using the linear regression equation of the standard curve.
Precautions
1. All fluorescent dyes are subject to quenching; minimize exposure to light to reduce fluorescence quenching.
2. PicoGreen working solution is best prepared fresh for optimal performance.
3. PicoGreen fluorescence intensity is highly temperature‑dependent; ensure standard curve construction and sample measurement are performed at the same constant temperature.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
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