PMC:3625153 / 31397-34248 JSONTXT

{"project":"2_test","denotations":[{"id":"23593491-15716106-88440279","span":{"begin":1610,"end":1612},"obj":"15716106"},{"id":"23593491-17083914-88440286","span":{"begin":2039,"end":2041},"obj":"17083914"},{"id":"T14208","span":{"begin":1610,"end":1612},"obj":"15716106"},{"id":"T4767","span":{"begin":2039,"end":2041},"obj":"17083914"}],"text":"Quantitative Real-Time PCR Analysis of ABCB and 5-HT1 mRNA Expressions\nTotal RNA was extracted from control and treated haemocytes using the ChargeSwitch total RNA cell kit according to the manufacturer's protocol. RNA concentration and quality were verified by UV spectroscopy and electrophoresis using a 1.2% agarose gel under denaturing conditions. First strand cDNA for each sample was synthesized from 1 µg total RNA in the presence of 250 ng random primers and 200 units RevertAid MulV reverse transcriptase following the manufacture's protocol.\nReal-time PCR reactions were performed in duplicate, in a final volume of 10 µL containing 5 µL Fast Sybr Green reaction mix with ROX, 2 µL diluted cDNA, and 0.2 µM specific primers (Table 1). A control lacking cDNA template was included in the real-time PCR analysis to determine the specificity of target cDNA amplification. Amplification was detected with a StepOne real time PCR system apparatus (Life Technologies, Milan, Italy) using a standard “fast mode” thermal protocol. For each target mRNA, melting curves, gel pictures and sequences were analysed in order to verify the specificity of the amplified products and the absence of artifacts. The amplification efficiency of each primer pair was calculated using a dilution series of cDNA (Table 1). A normalization factor, calculated using the geNorm software [56] and based on the expression levels of the best performing reference transcripts in the haemocyte samples, was used for accurate normalization of real-time PCR data. A set of suitable reference genes were selected from the literature [57]–[60] and are listed in Table 1; amongst these, the most stable reference genes used for normalization in haemocytes subjected to the different treatments were 18S rRNA and EF-α1 (Table 1).\n10.1371/journal.pone.0061634.t001 Table 1 List of primers used in real time PCR analyses. * primers were constructed basing on a partial sequence encoding an ABCB gene product from M. galloprovincialis (GenBank Ac Numb EF057747; [50]). A WWW-based database search using the BLAST program at NCBI found that this partial sequence showed a 65.17% and 64.93% nucleotide sequence identity with the human ABCB1 (GenBank Ac. Numb. NM_000927) and ABCB4 (GenBank Ac. Numb. NM_01884) gene sequences, respectively, while sequence identities lower or close to 50% were found with other human ABCB subtypes. Therefore, to avoid misleading information, the Pgp encoding gene from mussel investigated in this study will be referred to as ABCB gene. Relative expression of target genes in comparison with those of the reference genes was calculated by a comparative Ct method [61] using the StepOne software tool (Life Technologies, Milan, Italy). Data were finally reported as normalized relative expression (fold change) with respect to control samples."}