Real-time PCR: Instrument and Example


Bio-Rad CFX96 Real-time PCR Detection System
Bio-Rad CFX96 Real-time PCR Detection System

Example of real-time quantitative PCR

Figure A
amplification plot for real-time quantitative PCR experiment; Fig A

Figure B
standard curve for qPCR to determine sample concentration; fig b

An analysis was done of the mRNA levels for the tsaA gene (thiol-specific antioxidant protein) in the wildtype and a mutant strain of the bacterium Chlorobium tepidum (resides in hot springs of New Zealand).The mutant was created by insertional inactivation of the gene for rubisco-like protein. The tsaA product was measured with the dye SYBR Green, and standardized against the mRNA from the sigA gene (Sigma70 protein). Figure A shows the fluorescence of each well for each cycle, and Figure B shows the standard curve used to determine the concentration for the experimental samples.The mutant has a 13 fold increase in the tsaA mRNA level as compared to the wildtype which agrees well with the results found during a proteomics study of the same strains. The results are courtesy of Tom Hanson, Ph.D. (University of Delaware).

Example of High Resolution Melt Analysis

Example of High Resolution Melt Analysis difference curves

The Difference curves above derived from the melt curves are grouped based upon similarity and indicate that the two morphologically indistinct groups of mosquitoes are indeed different species based upon their differences in the 16s rRNA genes. Courtesy of Michael Zianni.