10. Check RNA Quality:
Assess aaUTP aRNA quality using the RNA 6000 Nano LabChip on the Agilent 2100 bioanalyzer according to manufacturers protocol (Figures 2a and 2b).
11. Fluorescent Dye Coupling:
Prepare NHS ester dye according to manufacturers protocol, using one of the following recommended commercial kits:
Amersham Biosciences CyDyePost Labeling Reactive Dyes
Perkin Elmer Cy Dyes
Molecular Probes Alexa Dyes.
12. Check Labeling Efficiency:
Measure 1L of labeled aRNA probe using the NanoDrop ND-1000 Spectrophotometer. Using a sample with sub-optimal frequency of incorporation (FOI) may result in low signal on the array, leading to loss of data.
12.1 Using the Microarray module of the NanoDrop software, select RNA-40 as the constant for measuring the aRNA component of the labeled probe. Ensure the appropriate dye type has been selected for measuring the dye component of the labeled probe.
12.2 The guidelines below can be used to determine if the FOI of the fluorescent dye is suitable for microarray hybridization. The example shows the use of Cy3 and Cy5 dyes:
Measure the absorbance of the fluorescent component of the labeled probe by selecting the appropriate dye from the drop down menu in the Microarray module. In this case, Cy3 a