Mapping of the transcription start site of MSTN gene–using SMARTer technology and 5'Rapid Amplification of cDNA Ends (5'RACE)

RNA was isolated as described previously. RNA was reverse transcribed to cDNA using the SMARTer RACE 5'/3' kit (TaKaRa-Clontech) which uses Switching Mechanism at the 5' end of RNA Template (SMART) technology and employs 5' Rapid Amplification of cDNA Ends (5'RACE) as per manufacturers’ instructions. The reaction mix was incubated for 90 minutes at 42°C followed by 10 minutes at 70°C in a hot-lid thermal cycler. Tricine-EDTA (10 μl) was then added to each reaction mixture and the 5'RACE ready cDNA was then either used immediately or stored at -20°C until use. The 5'RACE PCR was performed using SeqAmp DNA polymerase and associated buffer (TaKaRa-Clontech). The forward primer used in the reaction was the Universal primer mix supplied with the SMARTer RACE 5'/3' kit which binds to the universal primer binding sites incorporated during the SMARTer cDNA synthesis. The reverse primer used in the reaction was designed to specifically amplify the equine MSTN gene and was located within exon 1 of the MSTN gene. Both primer sequences contained a 15-nucleotide sequence which overlapped with a linearized vector for In-Fusion cloning of the fragment. Primers sequences were as follows: 5'-CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT-3' (forward), 5'GAT TAC GCC AAG CTT TCA GTT CCC GGA GTG GAG GAG CTT TGG-3' (reverse). A touchdown PCR program was employed, as follows; (A) 5 cycles of 94°C for 30 seconds and 72°C for 2 minutes, followed by (B) 5 cycles of 94°C for 30 seconds, 70°C for 30 seconds and 72°C for 2 minutes, followed by (C) 25 cycles of 94°C for 30 seconds, 68°C for 30 seconds and 72°C for 2 minutes. Once the PCR had finished a sample of the product was electrophoresed on a 1% agarose gel and imaged to determine the presence of product bands. If no bands or faint bands were apparent, the remainder of the PCR reaction, which was stored at 4°C during this time, was subjected to an additional 5 cycles of step C of the PCR program. The 5'RACE PCR products were electrophoresed on a 1% agarose gel and the DNA fragments of interest were located under UV light and were excised from the gel using a clean scalpel blade, as quickly as possible, to minimise the exposure time to the UV light and thus avoid damaging the DNA. The DNA was extracted from the gel using a NucleoSpin Gel and PCR Clean-Up Kit (TaKaRa-Clontech) as per manufacturers’ instructions. An In-Fusion HD Cloning enzyme master mix (TaKaRa-Clontech) was used along with a linearized pRACE vector (supplied with SMARTer RACE 5'/3' Kit). The In-Fusion cloning was performed as per manufacturers’ recommendations and the plasmid was transformed into Stellar^TM competent cells (TaKaRa-Clontech) and was grown at 37°C. Subsequently, the plasmid DNA was isolated and sequenced using M13 primers (5'-TGT AAA ACG ACG GCC AGT-3' (forward), 5'-CAG GAA ACA GCT ATG ACC-3' (reverse)) by MWG eurofins, to ascertain where the transcription start site was on the sequence, as the inserted fragment sequence would begin at the transcription start site, preceded by the universal primer sequence.