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The correlation between Ribo-Zero-Seq on FF and FFPE as well as between Ribo-Zero-Seq-FFPE replicate pairs has also been confirmed in Norton et al. [ 24].Lastly, we applied Deming regression to estimate a statistically unbiased slope to determine the relative sensitivity of protocol pairs.
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Although our experiments do not specifically address the question of detection limits for the ELISPOT and tetramer assays, we could detect a high variability in the sensitivity of protocols used by the different participants.
Since sensitivity of this protocol depends on the length of the amplified template, a suitable long extension qRT-PCR protocol was developed based on [72].
Synergy HT Microplate Reader used instead of Molecular Devices SpectraMax M5e Microplate Reader both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for sensitivity of detection (Protocol 1) and to determine if the gradient is similar to the original study (≤5%) (Protocol 2).
This results in a high sensitivity of the protocol for infiltrates and small nodular lesions.
Scanning the posterior thorax was added to increase the sensitivity of the protocol [23].
Secondly, all patients were hemodynamically unstable, so it must be assumed that the amount of bleeding was important, which could have improved the sensitivity of the protocol and partially compensate the inter-operator variability.
To improve the sensitivity of the protocol for mediastinal lymph nodes and bone lesions (e.g. metastases), a STIR or fat saturated T2-fast spin echo sequence should be added.
By applying the presented protocol to human lactoferrin, a widely studied glycoprotein, 6 new low-abundance glycans were discovered (unpublished data), which validated the sensitivity of the protocol.
The sensitivity of the protocol was determined after mixing a Jurkat T-cell clone in a background of peripheral blood CD4+ T-cells in a range of dilutions.
There are still variables that can be used to improve the sensitivity of the protocol, also when taking cost-effectiveness issues into regard.
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