The ineffectiveness, in the in situ procedure, of a primer that recognizes the 3' end of the mRNA, compared with the efficient use of one hybridizing upstream, is consistent with the interpretation that the ribosome is stalled at the end of the mRNA, which is consequently unavailable at the initiation of reverse transcription.
PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Inc .. Sequencing was carried out either with Cy5-labelled primers on an ALFexpress automated sequencer (Pharmacia) following standard procedures, or on a Beckman Coultier CEQ 2000 (Dye Terminator Cycle Sequencing) following standard procedures with the exception of primer concentration 5 μM instead of the recommended 1.6 μM.
To increase specificity of MSP procedure, four sets of primers were designed for promoter 1A of APC (NCBI accession No: U02509).
As a second modification of the procedure, the concentration of primers was increased from 2.5 ng/μl to 5-10 ng/μl.
Our optimization approach is a very common/well-known procedure whereby one studies combinations of primer concentrations in the range of 50 nM-900 nM while keeping the probe at a constant 200 nM, then the probe is challenged from 25 nM to 225 nM at the optimal primer concentrations established above.
See Supplemental Experimental Procedures for primer sequences.
The present work investigates the preparation of NiCe/γ-Al2O3 coated cordierite monoliths for oxidative dehydrogenation of ethane (ODE) reaction through a two steps procedure: the primer deposition of a γ-Al2O3 coating consisting in a mixture of micro and nanoparticles via the washcoating method and the latter incorporation of the active phase by impregnation.
For each procedure of bisulphite treatment, unconverted primers randomly covering the entire mtDNA molecule, including those used for the D-loop analysis, were used in polymerase chain reaction (PCR) reactions, as negative controls.
This procedure for sequence filtering opens up new possibilities for the optimization of primer design.
Results: Optimisations included RNA extraction procedure, design and concentrations of primers and probes from the 5′ non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn OAc 2 and thermolabile UNG concentrations.
