Description

picogreen assay

The PicoGreen assay is used to quantify the amount of DNA in a sample. Free, unbound dye does not fluoresce, but when bound to double-stranded DNA the dye fluoresces at a wavelength of 530nm. A plate reader quantifies the fluorescent intensity of the sample. To convert this into a concentration of DNA, a standard curve is prepared.

Within a given species, every nucleus contains the same DNA concentration, and most cells have only one nucleus. Therefore, the amount of DNA is directly proportional to the number of cells present. 

In this study, two polymers (E and M) are being investigated for their effect on cell growth in vitro. 36 wells were seeded with cells prior to incubation for 1 day (18 wells) or 7 days (18 wells). For each incubation period the wells were further subdivided into three groups of six. One group was considered the control (no treatment) while the other two groups were treated by the addition of porous polymers (E and M). Raw Data in fluorescence units (FU) are given below.

Day 1 FU Day 7 FU

Control Polymer E Polymer M Control Polymer E Polymer M

0.0785

0.1693 0.1935 0.1787 0.5171 0.3207

0.0858 0.1343 0.2037 0.1862 0.5005 0.3207

0.0814 0.1553 0.1876 0.1607 0.4751 0.3244

0.0825 0.1452 0.1791 0.1632 0.4751 0.3238

0.0802 0.1596 0.1889 0.1534 0.3991 0.4202

0.0798 0.1581 0.1921 0.1576 0.4226 0.4357

A standard curve was prepared using known concentrations of DNA, treated with PicoGreen, and fluorescence units were read.

DNA (ng/mL) FU

0 0.0020

12.5 0.2501

25 0.4802

50 0.8541

100 1.7503

Answer the following research questions:

Note: Use a significance level of α=0.05

Use linear regression to create a best fit model for the relationship between DNA concentration and FUs in the standard curve data. Include your R2 value here.

R2:

Create a figure of this model.

Use your model to convert FUs from the experiment to DNA concentration. Fill in the following table:

Day 1 DNA (ng/mL) Day 7 DNA (ng/mL)

Control Polymer E Polymer M Control Polymer E Polymer M

Perform a Two Factor ANOVA to compare Well Environment (Control vs. Polymer E vs. Polymer M) and Time (Day 1 vs Day 7). Include the p-values for Well Environment and Time factors here.

Well Environment p-value:

Time p-value:

Plot the change in DNA concentration over time for control, polymer E, and polymer M. Include error bars that represent the 95% CI on the mean. 

Place an asterisk in the correct cells to indicate statistical differences. 

Day 7 Control Day 1 Polymer E Day 7 Polymer E Day 1 Polymer M Day 7 Polymer M

Day 1 Control

Day 7 Control

Day 1 Polymer E

Day 7 Polymer E

Day 1 Polymer M

Verify the assumptions of equality of variance and normal distribution of residuals.

Instruction Files