You receive data back from the Qubit showing that your total RNA concentration i

Question

You receive data back from the Qubit showing that your total RNA concentration is 2.5 ug/mL. The sample run on the Qubit was diluted 100-fold, so you must correct for this to determine the actual concentration. For the same RNA sample, the Nanodrop gives you a value of 273 ng/uL.

1. How does the concentration from the Nanodrop compare to the concentration from the Qubit? SHOW your data and units conversions in your answer. Why do you think your concentrations from the Nanodrop and Qubit might be different?

2. If you need 1 ug of total RNA for the mRNA isolation step, what volume of your total RNA sample would you need (in uL) and which concentration should you use for this calculation and why?

Explanation / Answer

Quibit gave the reading of 2.5 ug/mL.

So the concentration of RNA sample will be 2.5 x 100 ug/mL = 250 ug/mL (since the sample was diluted 100 folds).

Unit conversion: 1 ug/mL = 1x (1000 ng/1000 uL) = 1 ng/uL.

Thus, Quibit gave the concentration = 250 ng/uL.

Nanodrop gave the reading of 273 ng/uL.

(1) From both the readings, there is a difference of 23 ng/uL (273-250 ng/uL). This can be due to error in dilution of the original sample. Although the values are comparable.

(2) As 1 ug RNA is required = means 1000 ng is required.

Considering the concentration to be (250+273)/2 = 261.5 ng/uL.

For 261.5 ng, volume required is 1 uL.

Therefore, for 1 ng, volume required will be 1/261.5

Thus, for 1000 ng, volume required will be (1x1000)/261.5 = 3.824 uL.

For calculating the volume, we used average of both the concentration, but it is better to use the lower concentration value in order to get enough RNA in the sample (otherwise, final amount of actual RNA may be less than 1 ug if higher concentration value is considered).

Considering, 250 ng/uL, we need (1x1000)/250 = 4 uL.

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