RNA was prepared from freshly dissected tissues with Trizol (Invitrogen) following the manufacturer's instructions, except that the isopropanol precipitation step was lengthened to overnight at -20°C and there were 2 ethanol washes instead of one. The cDNA samples were prepared using Superscript II (Invitrogen) and random primers, or iScript (BioRad) with random primers; a 5 min 65°C step was added to the iScript protocol before chilling and adding the enzyme. While GAPDH and actin samples were unaltered by the choice of random priming or oligo-dT priming, Kalirin transcripts, which are much longer, appeared to be 10-50-fold less abundant compared to GAPDH when using oligo-dT priming instead of random priming. Pairwise statistical comparisons used the t-test for two samples assuming unequal variance and the two-tailed value is reported.
