Total RNA was extracted from hippocampi, using NucleoSpin RNA II Kit (MACHEREY-NAGEL Inc., Bethlehem, PA), and quantified using a BioTek* Epoch* Microplate Spectrophotometer (Fisher Scientific Inc., Pittsburgh, PA). Real-time reverse transcriptase-PCR was used to quantify levels of IGF1 total mRNA and IGF1 mRNA variants, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Based on high homology, we designed primer and probe sets (Table 2), using Primer Express (Applied Biosystems, Foster, CA). All primers were confirmed by PCR and sequencing. Relative quantification of PCR products was based on value differences between the target and GAPDH control, using the comparative CT method (Taqman Gold RT-PCR manual, PE Biosystems, Foster City, CA). Samples were run in quadruplicate.

Primers for sheep IGF1 transcription start sites (TSS) in the hippocampus.

(outer) ACTGGCATCTTCACCTGCTT

(inner) CAAGAAATCACAAAAGCAGCACTT

(outer) GCAAGCACAGGGCCAGAT

(inner) GAAGAGATGCGAGGAGGATGT

(outer) AGGGATTTAGAGAAAATCCTCACATT

(inner) TATCTACAAAACACAGACACTGTAGA

mRNAs were extracted from normal newborn lamb hippocampus, lung, and liver, using NucleoSpin RNA II Kit and NucleoTrap® mRNA Mini Kit (MACHEREY-NAGEL Inc. Bethlehem, PA) and quantified, using a BioTek* Epoch* Microplate Spectrophotometer (Fisher Scientific Inc., Pittsburgh, PA). Extracted mRNA was subjected to 5′ RACE, using FirstChoice® RLM-RACE Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The primers used are shown in Table 2. The PCR products were cloned, and positive clones were sequenced, using DNAMAN Version 5.2 (Lynnon Biosoft, Quebec, Canada).

Genomic DNA was extracted from hippocampi. DNA was quantified, using standard spectrophotometry, and subjected to sodium bisulfite modification according to the manufacturer’s protocol (CpGenome DNA Modification Kit, Chemicon International, Temecula, CA, USA) to determine site-specific CpG methylation. Based on our 5′-RACE results, 12 CpG sites in the upstream region of the farthest transcription start sites (TSS) (about 667 bases) were selected for analysis. Two primer sets listed in Table 2 were used for PCR amplification (amplicon lengths of 260 and 350 bp, respectively).

ChIP was carried out with commercial antibodies that cross-reacted with sheep epitopes. We used antibodies directed against H3K9ac (Cell Signaling Technologies, Beverly, MA, USA), H3K14ac (EMD Millipore Corporation, Billerica, MA, USA), H3K4me3, H3K27me3, and H3K36me3 (Abcam, Cambridge, MA, USA), as described by our group.60 Primers and probes are listed in Table 2.

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