mRNA extinction coefficient and concentration calculator

PROTOCOL FOR ALKALINE HYDROLYSIS OF RNA
Materials:
- 1 part (e.g. 2 μl) of RNA stock solution.
- 1 part (2 μl) of same buffer as RNA stock solution.
- 10 parts (20 μl) of 1 M NaOH.
- 10 parts (20 μl) of 1 M HCl.
- 50 parts (100 μL) of nuclease-free water.

Procedure:
1) add 1 part (2 μl) of your stock RNA into a tube (#1),
2) add 4 parts (8 μl) of 1 M NaOH and incubate at 37°C for 1 hour or 20°C for 12 to 16 hours (overnight incubation) .
3) add 4 parts (8 μl) of 1 M HCl to neutralize the solution,
4) add 16 parts (32 μl) of nuclease-free water and mix to reach a 1/25 dilution of the stock RNA solution.
5) Prepare a black solution, by adding into a tube (#0):
   - 1 part (2 μl) of buffer solution,
   - 4 parts (8 μl) of 1 M NaOH,
   - 4 parts (8 μl) of 1 M HCl,
   - 16 parts (32 μl) of nuclease-free water.
6) Blank the UV spectrophotometer and measure the 260 nm UV absorbance,
perform 3 to 5 measurements, calculate the dilution corrected A260 mean value, using the A260 mean calculator.
7) Enter the RNA sequence.

8) Click on "Calculate".

Note:
Spectrophotometers are more accurate when the measurements are around 1 (0.8-1.2).
A serial dilution will assure that at least one of the measurements is within or close to this range.
Multiple measurements also help to reduce the impact of pipetting error on the mRNA concentration calculation.
An advanced protocol is here provided.
Materials:
- 4 part (e.g. 8 μl) of RNA stock solution.
- 15 part (30 μl) of same buffer as RNA stock solution.
- 100 parts (200 μl) of 1 M NaOH.
- 100 parts (200 μl) of 1 M HCl.
- 250 parts (500 μL) of nuclease-free water.

Procedure:
Perform steps 1 to 4 four times, i.e. in four different tubes (#1 to #4).
After step 4, prepare a diluting solution:
   - 11 parts (22 μl) of buffer solution,
   - 44 parts (88 μl) of 1 M NaOH,
   - 44 parts (88 μl) of 1 M HCl,
   - 176 parts (352 μl) of nuclease-free water.
Dilute tube #2 to 4, by adding 25, 75 or 175 parts (50, 150 and 350 μl) of diluting solution to reach 1/50, 1/100 and 1/200 dilution factors respectively.
Perform step 6 using tube #1-4.
These measurements allow the determination of the corrected A260 mean value using a Linear Regression model.
A Linear regression calculator is here provided.

Important notes:
- on tube selection:
PCR tubes or cap-locked tubes are recommended.
- on the 37°C incubation:
It can be performed on a thermocycler with cap-heating (105°C) capability or on dry heat block.
The reaction tubes should be cool-downed and centrifuged after this step to reduce evaporation.
- on the pathlength:
*Most comercially available microvolume spectrophotometers (a.k.a nanodrop) are calibrated for and use 1 cm as default pathlength.

PROTOCOL FOR THERMAL HYDROLYSIS OF RNA AT NEUTRAL PH
Materials:
- 1 part (2 μl) of RNA stock solution.
- 1 part (2 μl) of same buffer as RNA stock solution.
- 1 part (2 μl) of 10X hydrolysis buffer (0.6 M Na₂CO₃, 100 mM EDTA pH 8).
- 1 ml of nuclease-free water.

Procedure:
1) add 1 part (2 μl) of your stock RNA into a tube (#1),
2) add 1 parts (2 μl) of 10X hydrolysis buffer.
3) add 8 parts (16 μl) of nuclease-free water and and incubate at 95°C for 2 hours.
4) Prepare a black solution, by adding into a tube (#0):
   - 1 part (2 μl) of buffer solution,
   - 1 part (2 μl) of 10X hydrolysis buffer,
   - 8 parts (16 μl) of nuclease-free water.
5) add 15 parts (30 μl) of nuclease-free water to reach a 1/25 dilution of the stock RNA solution.
6) Blank the UV spectrophotometer and measure the 260 nm UV absorbance,
perform 3 to 5 measurements, calculate the dilution corrected A260 mean value, using the A260 mean calculator.
7) Enter the RNA sequence.

8) Click on "Calculate".

Note:
Spectrophotometers are more accurate when the measurements are around 1 (0.8-1.2).
A serial dilution will assure that at least one of the measurements is within or close to this range.
Multiple measurements also help to reduce the impact of pipetting error on the mRNA concentration calculation.
An advanced protocol is here provided.
Materials:
- 4 part (e.g. 8 μl) of RNA stock solution.
- 15 part (30 μl) of same buffer as RNA stock solution.
- 20 parts (40 μl) of 10X hydrolysis buffer,
- 400 parts (800 μL) of nuclease-free water.

Procedure:
Perform steps 1 to 5 four times, i.e. in four different tubes (#1-4).
After step 5, prepare a diluting solution:
   - 11 parts (22 μl) of buffer solution,
   - 11 parts (22 μl) of 10X hydrolysis buffer,
   - 253 parts (506 μl) of nuclease-free water.

Dilute tube #2 to 4, by adding 25, 75 or 175 parts (50, 150 and 350 μl) of diluting solution to reach 1/50, 1/100 and 1/200 dilution factors respectively.
Perform step 6 to 8 using tube #1-4.
These measurements allow the determination of the corrected A260 mean value using a Linear Regression model.
A Linear regression calculator is here provided.

Important notes:
- on tube selection:
PCR tubes are recommended.
- on the 95°C incubation:
it should be performed on a thermocycler with cap-heating (105°C) capability and reaction tubes should be centrifuged and cool-downed after this step to reduce evaporation.
- on the pathlength:
*Most comercially available microvolume spectrophotometers (a.k.a nanodrop) are calibrated for and use 1 cm as default pathlength.

A260 mean: Corrected A260 mean:

The result will be written in the "260 nm Absorbance (A260)" box

Calculate
The model is used to caculate the A260 value for your RNA stock solution.
The model-derived A260 mean value will be rounded at three decimal places and written in the "260 nm Absorbance (A260)" box.