Assessing the Quantity and Quality of the Purified cRNA

3.7.1. Assessing the Quantity of the Purified cRNA by Spectrophotometry

1. Turn on the absorbance spectrophotometer 15 min before use.

2. Calibrate the meter using a DEPC H2O control according to the machine's specific instructions.

3. Quantify RNA yield by spectrophotometric analysis at 260 and 280 nm for sample concentration and purity.

4. Dilute 1 |L of the cleaned cRNA in 4-9 |L of DEPC H2O (1:5 to 1:10 dilutions)

5. Measure the RNA concentration of each diluted cRNA.

6. Calculate the cRNA content in the undiluted sample multiplying by the dilution factor.

7. Apply the convention that 1 absorbance unit at 260 nm = 40 |g RNA (see Note 43).

3.7.2. Assessing the Quality of the Purified cRNA by Gel Electrophoresis

Gel electrophoresis of the IVT product is done to estimate the yield and size distribution of labeled transcripts. Follow steps 1 to 10 of Subheading 3.3.2. using cRNA instead of total RNA. In addition please perform an 11th step: View and photograph the gel under ultraviolet light using usual protective gear (see Note 44).

3.7.3. Assessing the Quality of the Purified cRNA Using the 2100 Agilent Bioanalyzer

Refer to the detailed and well-illustrated accompanying manual for further details to those below. For cRNA, the concentration range that can be quantified is between 25 and 250 ng/|L. Therefore, for most of your samples you will need to make a diluted aliquot.

1. Warm the reagents, including an aliquot of RNA 6000 ladder, to RT and a heating block to 70°C.

2. Turn on the Bioanalyzer and its workstation 15 min before use.

3. Start the Agilent 2100 Bioanalyzer software by double clicking on the Bioanalyzer desktop icon.

4. Prepare 3 ||L of each cRNA to be run with a concentration <500 ng/|L using DEPC H2O.

5. Denature the RNA samples a 3-|L aliquot of RNA 6000 ladder by heating to 70°C for 2 min.

6. Place denatured ladder and samples on ice (see Note 45). Decontaminating the Electrodes

1. Fill the first electrode cleaner chip via one of its wells with 350 |L of RNAseZAP.

2. Open the lid of the Agilent 2100 Bioanalyzer and insert the first electrode cleaner.

3. Close the lid; leave for 1 min and then remove the first electrode cleaner chip.

4. Add 350 |L of DEPC H2O to the second electrode cleaner chip.

5. Insert the second electrode cleaner chip in the Bioanalyzer and leave for just 10 s before removing.

6. Allow the Bioanalyzer electrodes to dry for 10 s before closing the lid. Loading the RNA Chip

1. Set the Chip Priming Station base plate to position C, insert the syringe (1 mL) into the syringe holder, and set the adjustable syringe clip to the highest position.

2. Place a new RNA chip on the chip priming station with the writing towards you and the 'cut off' corner top right.

3. Dispense 9 ||L of gel-dye mix (Subheading into the bottom of the 3rd well marked with a "G" from the top of the chip (see Note 46)

4. Raise the syringe plunger to the 1 mL mark and close the chip priming station.

5. Depress the plunger until it is held by the syringe clip.

6. Keep well "G" pressurized for exactly 30 s, then release the plunger and return it to the 1-mL mark.

7. Open the chip priming station and ensure that no bubbles are present in the chip channels by hold the chip to the light.

8. If bubbles are present in the channels, repeat steps 4 to 7 above (see Note 47).

9. Dispense 9 ||L of gel-dye mix into the bottom of the other two wells marked with a "G."

10. Pipet 5 |L of RNA 6000 Nano Marker (green dot on lid) into each sample well you will use and the ladder well.

11. Pipet 6 | L of RNA 6000 Nano Marker (green dot on lid) into each sample well not to be used.

12. Add 1 |L of denatured RNA 6000 ladder to the bottom of the well with the ladder symbol (bottom right).

13. Add a 1 |L of each denatured sample into each sample wells, loading from top left to bottom right (1 through 12; see Note 48).

14. Vortex the chip for 1 min at the recommended speed (IKA vortexer set-point)

15. Place the chip in the Bioanalyzer and close the lid. Begin the run within 5 minutes to avoid deleterious evaporation (see Note 49).

16. Return to the workstation, where the chip icon will now be displayed top left, indicating that a chip has been inserted and the lid successfully closed.

17. From the "Assay" menu, select "RNA" and then the type of RNA assay you wish to perform (usually Eukaryote Total RNA nano)

18. Click on the "Start" icon to bring up the "Start" dialog box.

19. Add your desired file prefix. The data will be saved to a file of this name.

20. Adjust the number of samples to be run and ensure that the "Edit samples after start" box is ticked.

21. Click on the "Start" button.

22. As desired, add the identifiers for each sample in the "Samples Information" window of the "General Chip Information" dialog box.

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