Densitometry was performed using a gel acquisition and analysis set-up (UV Products, Ltd .. Band densities were normalised to Hsc-70 to make quantitative measurements of the protein of interest.
Note that our specification that when cooperators dominate (p = 1), the equilibrium level of public good is normalised to one (ie e*(p = 1) = c/u = 1), entails c and u are expressed in units ensuring c = u.
Figure 3C shows the phase position of the SCN and retina by comparing the circadian variation in the average (n = 11 participants) melatonin concentration (black line; right ordinate normalised to one), intrinsic ipRGC post-illumination pupil response (blue line) and cone inputs to the ipRGC post-illumination pupil response (red line).
XIAP RNA levels were also normalised to two housekeeping genes (delta CT method), and were also occasionally compared to another QC sample acting as a reference point (delta delta CT).
The Ct values obtained from the qRT-PCR analysis were normalised to five housekeeping genes (beta glucuronidase [ Gusb], hypoxanthine quinine phosphoribosyl transferase 1 [ Hprt1], heat shock protein 90 alpha [ Hsp90ab1], glyceraldehyde-3-phosphate dehydrogenase [ Gapdh], and beta actin [ Actb]).
For microarray data, there was a good internal correlation between quantile normalised and non-normalised data for a given miRNA, however, the internal correlation between the relative expression by RT PCR of miR-210, and miR-210, normalised to three control genes, was noisier.
SNIP/p140Cap threshold cycle (CT) results were subsequently normalised to two suitable endogenous controls – β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) – and calibrated against a pooled cDNA from breast specimens, using the comparative CT method, 2−ΔΔ C T (Livak and Schmittgen, 2001).
The frequency of miRNAs in the two libraries was normalised to one million by the total number of miRNAs in each sample (transcripts per million (TPM) normalised expression = initial miRNA count*1,000,000/total count of clean reads).
