Nytran membrane

For protein analysis, 2-3x10⁶ cells were collected for each sample, resuspended in Laemmli buffer heated and subjected to SDS-PAGE gel electrophoresis. All assays of macromolecular biosynthesis and RNA processing were done at densities of less than 2 x 10⁶/mL. Pulse-labeling was done as described in [72 ].
Total RNA was extracted from roughly 5x10⁷ cells using peqGold TriFast (peqLab) following the manufacturer's instructions. The RNA was separated on formaldehyde gels and then blotted on Nytran membranes (GE Healthcare). Following crosslinking and methylene blue staining (SERVA), the northern blots were hybridized with the appropriate probes. For mRNA detection, the membranes were incubated with [α-³²P]dCTP radioactively labelled DNA probes (Prime-IT RmT Random Primer Labelling Kit, Stratagene) overnight at 65°C. For spliced leader detection, a 39mer oligonucleotide complementary to the spliced leader was labelled with [γ-³²P]ATP using T4 polynucleotide kinase (NEB) and incubated with the membrane overnight at 42°C. After washing the blot, it was exposed to autoradiography films and detection was performed with FLA-7000 (GE Healthcare). The images were processed with ImageJ.

Total RNA and poly(A+) RNA were isolated from brain tissues using the RNAeasy kit (QIAGEN), including deoxyribonuclease treatment, and the poly(A+) kit from Ambion (Now Life Technologies). Dio3 Northern analysis was performed following standard protocols. In brief, total and poly(A+) RNA samples were electrophoresed in a denaturing 1% agarose gel containing formaldehyde and blotted onto a Nytran membrane (GE Life Sciences). The blots were hybridized at 42°C in buffer containing 50% formamide, washed with 0.1× saline sodium citrate/0.1% sodium dodecyl sulfate at 65°C and autoradiographed for 1–7 days. Probes were labeled with radioactive ³²P-dCTP (MP Biomedicals, Inc) using the oligolabeling kit (Pfizer) and were purified through G-50 columns (Pfizer). A 1.35-kb XhoI restriction fragment comprising the Dio3 coding region and part of the 3′-untranslated region was used as a probe.
DNA was isolated from tail snips using a kit from QIAGEN. A Southern analysis was performed using standard protocols on genomic DNA (gDNA) digested with EcoRI. A 0.7-kb SacI/EcoRI restriction fragment located immediately outside the 5′ end of the targeting sequencing was used as a probe.