Refer to the detailed and wellillustrated accompanying manual for further details to those below For total RNA the concentration range that can be quantified is between 25 and 500 ngL

1. Therefore, for most of your samples you will need to make a diluted aliquot. 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. Start the Agilent 2100 Bioanalyzer software by double clicking on the Bioanalyzer desktop icon.

3. Prepare 3 |L of each total RNA to be measured with a concentration <500 ng/|L using DEPC H2O.

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

5. Place denatured ladder and samples on ice (see Note 21).

3.3.3.1. 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 Bioanalyser electrodes to dry for 10 s before closing the lid.

3.3.3.2. 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 into the bottom of the third well marked with a "G" from the top of the chip (see Note 22).

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 23).

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 24).

14. Vortex the chip for 1 minute 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 25).

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" dialogue box (see Notes 26 and 27).

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