Summary+for+search+indexing+10-2-12

Summary: Tumor Library Lysate 10-2-12 Selected tumor library lysates were probed with tumor and naïve sera at 3 different dilutions: 1:1000, 1:500, 1:100. This experiment was a repeat of the two previous experiments which gave some unusual results. In the previous experiment, analysis of the means of the slides revealed a very clear pattern that should not have been present.

The mean intensity of each slide was steadily decreasing. I suspected that this was a poor Tecan run at the time since the slides were not dried before use, and each chamber had liquid above the hole before the sample was added. Therefore, each addition of blocking buffer and sample was diluted by some unknown amount. The analysis of the previous experiment also revealed that the naïve was higher than the tumor for all but one sample when they all should have been identical, which further supported the inconsistency of this experiment. From the previous two experiments, I also found that the naïve sera had a lower intensity than the tumor at a 1:500 dilution, but that the naïve sera had a higher intensity than the tumor at a 1:100 dilution. This was the opposite result of my original expectations. However, I now hypothesize that the naïve sera has more total antibody in the sera and therefore there is more non-specific binding when the concentration is increased. In this experiment, I tried three different dilutions. The slide means of this experiment do not reveal any consistent pattern other than the change in total intensity from different slide dilutions as would be expected.

This experiment revealed that the tumor sera had higher intensity for the spots than the naïve sera at higher dilutions as I predicted from the previous experiment. Also note that all 12 of my spots that are not e coli spots potentially bind to the tumor sera more than the naïve because these spots were selected for high tumor sera binding. In this experiment, many of the selected tumor lysate spots had higher intensity with tumor sera than naïve with high fold changes and low p values, whereas the e coli spots did not meet these criteria. Note that the Tecan had a “low pressure” error during this experiment, and we believe that the compressor failed for reasons unrelated to this particular experiment. The compressor just needs to be maintained. However, this error does not seem to have affected most of my slides in any way.

Comparison of Naïve and Tumor Sera Intensities for Selected Tumor Library Lysates

Here is the 1:1000 sera graph with a different scale since the more dilute sera had lower intensities as expected.

The 1:1000 slides were scanned again at a higher laser power since they had a lower intensity and analyzed further.

Here are the p values and fold changes of the lysates for the 1:1000 sera. The data is sorted first by fold change and then by p value. Note that some of the “Empty” samples do have a significant p value between naïve and tumor. Part of the reason for this is that the background on the slide caused by the different sera types is slightly different. However, the fold change between the background intensity with the tumor sera and the naïve sera is small. The p values are significant because there are many replicates.

Name p value fold change 11 0.120641 1.496796973 41 0.138791 1.329429942 4 0.074533 1.312103352 42 0.040345 1.294987794 62 0.234767 1.288551105 21 0.103861 1.287529591 33 0.002274 1.284193801 81 0.322135 1.275062232 71 0.066797 1.260217273 31 0.014972 1.249209009 Ecoli 0.426325 1.217563964 51 0.145466 1.18519441 61 0.338157 1.161101503 Empty 0.002428 1.119325487 Empty 0.040747 1.116498886 Empty 0.329361 1.056869089