Fluorescence Information and Courses from MediaLab, Inc.

After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry.An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity. The densitometer scan below depicts the separated bands from a serum sample electrophoresis. The SPIFE 3000, Helena Laboratories, electrophoresis splits the beta zone into two fractions for easier detection of small beta-migrating monoclonal gammopathies. The densitometer scan from this electrophoresis shows five bands with two peaks in the beta band.

Molecular diagnostic techniques utilize CE extensively. Automation, microvolume sample, increased sensitivity, immediate detection, and the computerization provided by CE enhance the analysis of nucleic acids. A multiple fluorescence detection system available with CE is also valuable.CE analysis of short tandem repeat polymorphisms is used in forensics, parentage testing, bone marrow engraftment analysis and other identification assays. Other testing for diagnosis of genetic diseases, oncology studies and DNA sequencing frequently utilize CE. DNA sequencing uses CE for separation of nucleotides labeled with multiple colored fluorescence dyes; CE and these markers enable computerized determination of the nucleotide sequence of DNA segments.

Some global specimen collection and handling issues to consider include: Specimens that contain nucleated cells will be of interest in DNA methodologies while specimens lacking nucleated cells are more useful in RNA methodologies. rRNA is more stable than mRNA, which is labile and sensitive to contamination. DNA is relatively stable and can be obtained from nonviable sources. Serum or plasma obtained by standard routine venipuncture procedures can be used as long as proper site selection and decontamination occur. Standard anticoagulants such as Ethylenediaminetetraacetic Acid (EDTA) and Acid Citrate Dextrose (ACD) can be used; however avoid the use of heparin as an anticoagulant as it interferes with some polymerase chain reaction (PCR) methodologies. When using fluorescence, fasting serum or whole blood specimens should be used to decrease the interference by lipids.

Detection techniques can vary in both direct and amplified methodologies and can include labeling either the probe or the target molecule of interest:Chemiluminescence: Release of light energy at the end of a chemical reaction that is detected by a luminometer. Uses a label such as acridinium ester. Electrophoresis: movement in a matrix such as a gel that is caused by an electrical field.Enzyme: Uses enzyme and substrate principles to label the appropriate target or probe. Can be combined with fluorescence or dyes for detection.Fluorescence: Molecules that emit light at a longer wavelength when excited at a shorter wavelength. Detection techniques include fluorescent staining of nucleic acids as well as fluorescent labeled probes that are measured in a fluorometer or with fluorescent polarization.Radioactivity: Uses a labeling technique where the radioactive label is then measured in a scintillation counter. The earliest assays utilized radioactive decay.