Random priming method

Total RNA was isolated from cells using RNA STAT-60 reagent (Tel-Test, Friendswood, TX) following the manufacturer’s instruction. An amount of 15 μg of total RNA was denatured and electrophoresed on 1% agaro-seformaldehyde gels. The uniformity of sample loading was verified by UV visualization of the ethidium bromide-stained gel before transfer to Nylon membrane. ThecDNAprobes for MCPIP1, 2, 3 and 4 were amplified by PCR using individual cDNA clones from ATCC as templates. 32P-labeled cDNA was prepared using the random priming method (Invitrogen). Hybridization was performed using QuickHyb buffer (Stratagene) at 65°C for 2 h or overnight. The membranes were then washed once with 2XSSC and once with 0.1_SSC, 1% SDS for 20 min at 65°C.

Total RNA was isolated from mouse tissues using RNA STAT-60 reagent (Tel-Test) following the manufacturer’s instruction [11 (link)]. Fifteen micograms of total RNA was denatured and electrophoresed on 1% agarose-formaldehyde gels. Uniformity of sample loading was verified by UV visualization of ethidiumbromide-stained gels before transfer to Nylon membranes. The cDNA probes for PAMM were amplified by PCR using a cDNA clone from A.T.C.C. as templates. ³²P-labelled cDNA was prepared using the random priming method (Invitrogen). Hybridization was performed using QuckHyb buffer (Strategene) at 65°C for 2 h or overnight. Then membranes were washed once with 2 × SSC and once with 0.1 × SSC, 1% SDS for 20 min at 65°C.