Fluorescent Dyes Design and Protocols

Fluorescent Dyes Design / Protocol

A variety of fluorescence-based systems can be used to analyze samples using RT-qPCR, including sequence-specific nucleic acid probes (ex: 5’-nuclease probes, molecular beacons, FRET hybridization probes, Scorpions) and non-sequence-specific fluorescent intercalating dyes (ex: SYBR Green, Cyto dyes). No matter which system you choose, however, it is critically important that the proper negative and positive controls be run to ensure that your experimental results can be correctly interpreted. The following controls should be part of every RT-qPCR experiment.

a) a no-reverse-transcriptase control: For this control, the reverse transcripase enzyme is not included in the reverse transcription reaction. Performing this control allows you to determine the amount of genomic DNA contamination in your RNA preparation.

b) a no-template control: For this control, the cDNA template is not included in the PCR reaction. Performing this control allows you to determine the amount of nucleic acid contamination and/or primer-dimer formation. It is particularly important to do this control if you are using a fluorescent intercalating dye for detection.

c) a no-amplification control: For this control, the DNA polymerase is not included in the PCR reaction. Performing this control allows you to determine background fluorescence in the PCR reaction.

a) exogenous positive control (run in a separate tube): For this control, external RNA or DNA, containing the target of interest (but NOT one of your experimental samples), is added to the reverse transcription or PCR reaction in place of an experimental sample. This control allows you to verify that the reaction is working properly, that the reaction is not being inhibited by something in the reaction mix, and that the fluorescence signal is being generated and detected. Although PCR-generated and purified amplicons can be use as exogenous positive controls, using a synthetic ultra-long oligo (Synthetic Positive Control –SPCT) is more robust because it provides you with a known copy number.

b) endogenous positive control (can be run in a separate tube or in multiplex with the experimental sample): For this control, a second target, native to the species of interest and known to be in the experimental sample, is used to normalize the various fluorescent signals obtained from those samples. Most commonly, reference genes are selected for this. Such genes are ubiquitously expressed in all tissues. For example, a list of such reference genes for mouse is found here, and for human, here.

References

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(3) Giusti, W.G., Adriano, T. Synthesis and characterization of 5'-fluorescent-dye-labeled oligonucleotides. Genome Res. (1993), 2: 223-227.
(4) Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations. Hum. Mutat. (1995), 5: 153-165.
(5) Fan, J.B., Chen, X., Halushka, M.K., Berno, A., Huang, X., Ryder, T., Lipshutz, R.J., Lockhart, D.J., Chakravarti, A. Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays. Genome Res. (2000), 10: 853-860.
(6) Didenko, V.V. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Design and Applications. Biotechniques (2001), 31: 1106-1121.