The fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify mRNA level in cells and tissues and is now a crucial tool for basic biological researches and biotechnology. In the present study, on the basis of the real-time quantitative RT-RCR, we detected and quantified mRNA copies of the transcription factor, CCAAT/enhancer binding protein (C/EBP; an immediate-early gene that is involved in synaptic plasticity and learning and memory) in the central nervous system of the pond snail Lymnaea stagnalis. We designed the primer set and the probe in the specific insert for the detection of Lymnaea C/EBP (LymC/EBP) clone 1. This insert is not contained in LymC/EBP clone 2 by alternative splicing. The copy number of LymC/EBP clone 1 was linearly decreased relative to the dilution of cDNA, and it was estimated 30 copies/ml in test sample. The availability of the present study showed that the real-time quantitative RT-PCR technique is more accurate and more specific for the detection and quantification of the mRNA level of genes in L. stagnalis than the other PCR methods.
Authors:S. Naeimi, S. Kocsubé, Zsuzsanna Antal, S. Okhovvat, M. Javan-Nikkhah, C. Vágvölgyi and L. Kredics
In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.
Authors:Sobiya Shafique, Shazia Shafique and Aqeel Ahmad
gene due to its strong recommendation by Coker and Davies [ 22 ]. A primerset used for studying α-Tubulin expression was the same as used by Xu and Shi [ 23 ], at their recommended thermocyclic conditions in GeneAmp-2700 thermocycler (Applied
Authors:Markus Krohn, Thomas Wanek, Marie-Claude Menet, Andreas Noack, Xavier Declèves, Oliver Langer, Wolfgang Löscher and Jens Pahnke
photometrically. cDNA was synthesized using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, USA). Gene expression was analyzed with qRT-PCR using TaqMan Hybridization Probes. Primersets with according TaqMan probes for Abcb1a (Mm00440761_m1), human ABCB