Gc rich pcr system

PCR was performed as per manufacturers’ instructions using the GC-RICH PCR System (Roche Applied Science). PCR was performed using an initial denaturation for 3 min at 95°C followed by 10 cycles of: 30 s at 95°C, 30 s at 65°C, 45 s at 72°C; then 25 cycles of: 95°C, 30 s at 65°C, 45 s (or 1 min depending upon the amplified region) at 72°C; a final elongation for 7 min at 72°C, and then stopped at 4°C. Primer sequences are listed in Table S1 (ST1).

RT-PCR analysis was performed with the following RET primers: RET225 5′-GGCTGCGTCTGCTGTGC TG and RET466 5′-AAGGTGAGGAGGCCGGTGTC, covering sequences encoding extracellular parts, RET 2185 5′-GCCCACAGCCACCCAT and RET 2469 5′-GGA GGCGTTCTCTTTCAGCATC, covering transmembrane encoding parts, RET2738 5′-TGGGCGACCTCATCTC ATTTG and RET3271 5′-AGGCCGTCGTCATAAATC AGGGAG, covering the tyrosine kinase encoding sequences of RET mRNA. For the analysis of ligand transcripts, the GDNF specific primers GDNFfwd 5′-ATGTCGTGGCTGTCTGCCTGG and GDNFrev 5′-CATCGCAAGAGCCGCTGCAG, the PSPN specific primers PSPN90fwd 5′-CGTGGCCGATGGAGAGTT CTC and PSPNrev 5′-AAGGCCACGTCGGTGTAGCG, the NRTN specific primers NTNfwd 5′-AGAGGGCC TGCTTCTCAGCC and NTNrev 5′-TAGCGGAACAGC ACCGTCTCG, and the ARTN specific primers ARTNfwd 5′-TGCTGAGCAGCGTCGCAGAGG and ARTNrev 5′-AGGAGCCGCTGCAGAAGCGG, were used. To avoid amplification of contaminating genomic DNA, all primer pairs were designed to amplify cDNA fragments that spanned over one or several exon borders. Unigene representative sequences were used as template for primer design. PCR was run for 35 cycles using a GCRICH PCR system (Roche Diagnostics) and AmpliTaq GOLD Polymerase (Perkin-Elmer) according to the manufacturer's recommendations. PCR detection of FUSDDIT3 fusion transcripts was performed as previously described [4 (link), 56 (link)].

The c.585delG variant was analyzed on cDNA using Restriction Fragment Length Polymorphism (RFLP). The primers used (Table S2) were designed on the rabbit MLPH gene sequence (ENSOCUG00000016496) with Primer3plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plusAbout.cgi). PCR was carried out using a 2720 thermal cycler (Life Technologies) with the GC-Rich PCR System (Roche) in a 25-µL reaction volume containing 2.5 µL cDNA dilution in 1/5, 1 U GC-rich enzyme, 1X buffer, 0.2 mm dNTPs, 0.4 µm of each primer and 1.5 mm MgCl₂. The PCR profile was as follows: 5 min at 95 °C, 45 amplification cycles of 30 s at 95 °C, 30 s at 56 °C, 30 s at 72 °C, 10 min at 72 °C. Digestion was performed with SmlI enzyme (New England Biolabs) in a 12-µL reaction volume containing 4 µL PCR product, 1 U enzyme, 1X buffer at 55 °C overnight. The digestion products were electrophoresed on 2% agarose gel and were visualized with bromide of ethidium. In animals that were homozygous wt/wt for the c.585delG variant, a 173-bp undigested fragment was expected, while in rabbits homozygous mut/mut, digestion by SmlI was suggested, leading to two fragments of sizes 123 and 50 bp.

Because of its high GC content BICCprom was amplified using BICC1_Long_Prom_F and R oligonucleotide primers (Table 1) from human placental DNA using a GC-rich PCR system (Roche, East Sussex, UK). BICC77 was amplified using ECR rs2393477_F and R oligonucleotide primers (Table 1) from human placental DNA using the Expand High Fidelity PCR system (Roche). PCR products were cloned into pGEM-T-easy (Promega, Southamton, UK) and sequenced to confirm PCR fidelity and to determine genotype. To reproduce both haplotypes of BICC77 site-directed mutagenesis of BICC77 in pGEM-T Easy was undertaken using BICC1-SDM-61 primers (forward and reverse) and BICC1-SDM-77 (forward and reverse; Table 1) oligonucleotide primers using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Edinburgh, Scotland, UK).

An IGFBP2 mammalian expression construct was created and stably tranfected into OE33 cells. IGFBP2 was PCR-amplified from the cDNA of endogenously expressing Flo-1 cells using the GC-Rich PCR System (Roche, Indianapolis, IN) and primers containing EcoRI and BamHI restriction sites for directional cloning into the pEGFP-C1 vector (Clontech, Mountain View, CA). The pEGFP-IGFBP2 or empty vector construct was transfected into OE33 cells using FuGENE 6 transfection reagent (Roche) per the manufacturer's instructions and selected with 1000 μg/mL Geneticin (Invitrogen). Colonies were ring-cloned, expanded and then maintained in growth media containing 200 μg/ml Geneticin.