Publication protocol
An equal amount of RNA (1 μg) was used to prepare cDNA [17]. Complementary DNA was performed with Superscript II reverse transcriptase (ThermoFisher, Germany), 5× first-strand buffer (Thermo Fisher Germany), DTT (Invitrogen, Germany), dNTPs (GE Healthcare, Germany), linear acrylamide (Ambion, Germany), hexanucleotide (Roche, Germany) and RNasin (Promega, Germany). Reverse transcriptase reaction was performed for 90 min at 42 °C then the reaction was heated at 85 °C for 5 min using a Mastercycler pro (Eppendorf, Germany). RN-related molecule mRNA expression in human and mouse solid organs, as well as diseases model, were quantified by RT-PCR using GAPDH as housekeeper gene for human samples and 18 s for mouse samples as described previously [16]. Each PCR reaction (20 μl) involved 10 × Taq Polymerase Buffer, Taq Polymerase, dNTPs, BSA, PCR Optimizer, SYBR green dye, MgCl2, gene specific primers and 0.2 μl of synthesized cDNA. SYBR Green Dye fast-two step detection protocol from Light Cycler 480 (Roche, Mannheim, Germany) running program was used for amplification. Each amplification step included initiation step 95 °C, annealing step 60 °C and amplification step 72 °C and was repeated 40 times. Gene-specific primers (300 nM, Metabion, Martinsried, Germany) were used as listed in Table 1. DdH2O was used as negative control for target and housekeeper genes. A specific primer for each target was designed using the primer designing tool (NCBI) and In silico specificity screen (BLAST) was performed. The lengths of amplicons were between 90 and 120 bp. The kinetics of the PCR amplification (primer efficiency) was calculated for each set of primers. The efficiency-corrected quantification was performed automatically by the Light Cycler 480 based on extern standard curves describing the PCR efficiencies of the target and the reference gene [ratio = Etarget ΔCPtarget (control − sample)/Eref ΔCPref (control − sample)]. The high confidence algorithm was used to reduce the risk of the false positive crossing point. All the samples which rise above the background fluorescence (crossing point Cp or quantification cycle Cq) between 5 and 40 cycles during the amplification reaction were considered as detectable. The melting curves were analyzed for every sample to detect unspecific products and primer dimers. To visualize the similarity and differences in gene expression profiles among the samples, hierarchical cluster analysis were performed using algorithms incorporated in the open-source software MultiExperiment Viewer (MeV) version 4.9 [25]. Differentially expressed mRNAs were screened by Volcano Plot between log(2)(fold change) gene expression [unstandardized signal] against -log(10)(p-value) from the t-test [noise-adjusted/standardized signal] [26].
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