We did not detect statistically significant gains in other components of experimental design, such as identification of experimental system, treatment, assay, quantity measured, identification of controls, and statement of anticipated outcomes.
The experimental design score was based on seven components that included appropriateness (the match between the hypothesis and the proposed experiment), identification of experimental system, treatment, control group, assay, quantity measured, and expected outcomes (Supplemental Table S3).
Influx activity was stopped by washing with 500 µl of ice-cold 1 mmol/L TC and cells lysed with 250 µl of RIPA buffer (Sigma-Aldrich Gillingham, UK), 175 µl of which was mixed with 2 ml scintillation cocktail for radioactivity counting and the remainder used to assay protein quantity (Pierce BCA protein assay kit, Cramlington, UK) for normalisation.
Statistically significant gains were observed in "experimental system," "independent variable/treatment," "assay," and "quantity measured" components of experimental design (Supplemental Figure S5).
For instance, Takashi's group reported that lncRNA Air interacted with the H3K9 histone methyl-transferase G9a by CLIP [ 57], but their data suggest that the post-assay RNA quantity is small and the assay is tedious steps.
For most assays 20 ng of cRNA was injected, but for [C] kinetic assays, injection quantity was adjusted to ensure that uptake was linear with time.
Sensitivity assays comparing quantity of GHA DNA detected between pure and mixed samples showed that background genomic DNA from bark and leaf did not significantly affect detection sensitivity.
For the hemizygous experiments and the competition assay, equivalent quantities of mock-transfected cells seeded in 24-well plates were used to subtract the background fluorescence.
Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile.
To test the technical replicacy of the assay, different quantities of the same cell lysates were analyzed using two pairs of interactions (NF-κB1/RelA and TRB3/FN1).
Amplified products were column-purified with the QIAquick PCR Purification Kit (Qiagen, Dusseldorf, Germany) and assayed for quantity and quality with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
