I would like to confirm if there are any issues with the 5200 Fragment Analyzer instrument

Question

Hi 
 I used promega RNA ladder to confirm the instrument status (Figure 1). Is the difference in size (nt) within a reasonable range? (Figure 2) 
 The concentration in Figure 3 should be twice as high as in Figure 4, but the reason for the lower concentration observed in Figure 3 is what? In Figure 5, the concentration should be similar to Figure 6, but the situation presented in Figure 5 shows no signal. What could be the possible reasons? 
 Additional information: The RNA ladder I used is Agilent DNF-472 HS RNA (15nt) 
 Thank you 
 Figure1 
 
 figurre 2 
 
 figurre 3 
 
 figurre 4 
 
 figurre 5 
 
 figurre 6

Cendy.D · Answer

Hi Calvin, 
 Thank you for your post. 
 It appears that you used kit DNF-472. This kit has a 20% sizing accuracy specification. Overall, from Figure 2 shared, it appears that sizing is within 20% of the expected bands (Figure 1). 
 I cannot determine the reported concentration of the sample from Figures 3 and 4, however, issues with reported concentration may be related to using incorrect reagents, incorrect method, high salt content in samples, overloading samples above the quantitative range of the kit, insufficient mixing (must mix samples as indicated in the HS RNA Kit - DNF-472 Quick Guide), or an array requiring conditioning. 
 Figure 5 appears to have the lower marker present, however, sample is not present. A possibility is that the sample was inadvertently not added to the well. 
 If you require further assistance, I recommend contacting us at electrophoresis@agilent.com . Please share the raw data files as these are most informative and allow us to have visibility to various features including but not limited to capillary positions, migration and separation parameters of the run. To generate this, open the data file in ProSize and go to Help>Zip Opened Data file. All the required raw data files will be saved in a zip file in the location of the original data. Send in this .zip file.