RNA extraction and cDNA synthesis Total RNA was prepared using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. RNA was treated with RNase (Invitrogen) in the presence of 50 μM T7 (dT12) AP1, T7 (dT12) Vactosertib cell line AP5 and T7 (dT12) AP8 primers in 20 μl RT buffer (1× Superscript II RT buffer, 10 mM DTT, 0.025 mM dNTP), at 25°C for 5 minutes, followed by 50°C for 50 minutes. Reverse transcriptase was inactivated at 70°C for 15 minutes. Differential display and full-length

gene selleck chemical cloning Differential display was performed using Hieroglyph mRNA Profile Kit (Beckman, CA, USA). Briefly, PCR amplification was done using 1.5 μl of the cDNA, primed with arbitrary P primer and anchored T primer. Amplification at (95°C 2 minutes) 1 cycle, Selleck RAD001 (92°C for 15 seconds, 50°C for 30 seconds, 72°C for 2 minutes) 4 cycles, (92°C for 15 seconds, 60°C for 30 seconds, 72°C for 2 minutes) 30 cycles, followed by a final extension at 72°C for 7 minutes on a GeneAmp PCR system 9600 (Perkin-Elmer, Norwalk, USA). Following amplification of randomly primed mRNAs by RT-PCR, the cDNA products were heated at 95°C for 2 minutes and separated on a denaturing 5.6% polyacrylamide gel at 55°C for 5 hours using

a Genomyx LR DNA Sequencer (Beckman), under 3000 V. Bands exclusively present in either of two samples were considered as candidates of differentially expressed transcripts, which were excised, eluted, re-amplified, and subcloned into the T easy vector (Promega, San Luis Obispo, CA, USA). The sequence reactions were performed by Invitrogen. Sequence homology to published database was analyzed with the BLAST program at the internet site of NCBI (National Center for Biotechnology Information). 5′-RACE (rapid amplification

of cDNA 5′ ends) and 3′-RACE were used to isolate the complete cDNA. The human Marathon-ready cDNA (Clontech, Heidelberg, Germany) served as the template. Real-time quantitative reverse transcription polymerase chain reaction We measured LCMR1 gene expression in 95C and 95D cell lines by real-time quantitative RT-PCR in an ABI PRISM 7500 Sequence Detection System. The real-time RT-PCR allows, by means of fluorescence emission, the identification of the cycling point when PCR product is detectable. The Ct value inversely correlates with the starting Histidine ammonia-lyase quantity of target mRNA. Measurements were performed in duplicate and the controls were included in which the reaction mixture contained no cDNA. The amount of target mRNA after normalized to the loading control β-actin was calculated by the Ct method. Primers for β-actin and LCMR1 mRNAs were chosen using the Primer Express 2.0 software (Applied Biosystems, Foster City, USA). Primers for LCMR1 were: 5′-AACAGAGCCGTACCCAGG AT-3′ (Forward) and 5′-GGGTGGTCTGGACATTGTC -3′ (Reverse). Primers for β-actin were: 5′-CATGTACGTTGCTATCCAGGC-3′ (Forward) and 5′-CTCCTTAATGTCACGCAC GAT- 3′ (Reverse).