Magnapure mrna isolation kit

From cells, mRNA was isolated with the MagnaPure-LC device using the mRNA-I standard protocol. Tissue samples were disrupted with RiboLyser devices (ThermoHYBAID, Heidelberg, Germany) containing 400 μL lysis buffer from the MagnaPure mRNA Isolation kit containing 1% DTT (v/w) (ROCHE Diagnostics, Mannheim, Germany). mRNA was isolated with the MagnaPure-LC device using the mRNA-standard protocol for cells. mRNA was reversely transcribed using AMV-RT and oligo-(dT) as primer (First Strand cDNA synthesis kit, Roche, Mannheim, Germany) according to the manufactures protocol. Primer sets optimized for the LightCycler (RAS, Mannheim, Germany) were purchased from SEARCH-LC GmbH (http://www.search-lc.com/). The PCR was performed with the LightCycler FastStart DNA Sybr GreenI kit (RAS) according to the protocol provided. To control for specificity, a melting curve analysis was performed. The copy number was calculated from a standard curve, obtained by plotting known input concentrations of four different plasmids at log dilutions to the PCR-cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. To correct for differences in the mRNA content, the transcript numbers were normalized according to expression of the housekeeping gene peptidylprolyl isomerase B (PPIB). Values were given as transcripts per 1000 transcripts of PPIB.

The tissue samples were disrupted with RiboLyser devices (ThermoHYBAID, Heidelberg, Germany) containing 400 μl lysis buffer from the MagnaPure mRNA Isolation Kit containing 1%DTT (v/w) (ROCHE Diagnostics, Mannheim, Germany). mRNA was isolated with the MagnaPure-LC device using the mRNA- standard protocol for cells. An aliquot of mRNA was reversely transcribed using AMV-RT and oligo- (dT) as primer (First Strand cDNA synthesis kit, ROCHE Diagnostics, Mannheim, Germany) according to the manufactures protocol in a thermocycler. Primer sets optimized for the LightCycler® (RAS, Mannheim Germany) were purchased from SEARCH-LC GmbH (
http://www.Search-LC.com). The PCR was performed with the LightCycler® FastStart DNA Sybr GreenI kit (RAS) according to the protocol provided. To control for specificity, a melting curve analysis was performed. The copy number was calculated from a standard curve, obtained by plotting known input concentrations of four different plasmids at log dilutions to the PCR-cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. To correct for differences in the content of mRNA, the calculated transcript numbers were normalized according to the expression of the housekeeping gene peptidylprolyl isomerase B (PPIB). Values were thus given as transcripts per 1000 transcripts of PPIB.