Mezclas maestras para qPCR SYBR™ Green

Sensitivity can actually be equivalent when using TaqMan chemistry and SYBR Green reagent chemistry. It might seem that a TaqMan assay with fluorescent signal generated by a sequence-specific probe would always be more sensitive than a SYBR Green reagent assay, but a poorly designed TaqMan assay could theoretically be less specific than a well-designed SYBR Green reagent assay. However, the potential for detection of primer dimers and non-specific products using SYBR Green chemistry is more likely to result in loss of sensitivity when attempting to quantitate lower copy numbers.

For more information on Real-Time PCR chemistries, please refer to the following Application Notes, which you can find on our website through Technical Resources, or by entering the titles in the main Search field: “Real-Time PCR Vs. Traditional PCR”, “Essentials of Real Time PCR”, and “Selection of Reagents for Real-Time PCR”.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

The TaqMan MGB probes contain the following features:
1) A fluorescent reporter at the 5' end
2) A nonfluorescent quencher at the 3' end. Because the quencher does not fluoresce, the real-time instruments can measure the reporter dye contributions more precisely.
3) A minor groove binder at the 3' end. The minor groove binder increases the melting temperature (Tm) of probes, allowing the use of shorter probes.

In general, the TaqMan MGB probes exhibit great differences in Tm values between matched and mismatched probes, which provides more accurate allelic discrimination and makes for a more sensitive real-time assay. Mismatches between a probe and allele, or target, reduce the efficiency of probe hybridization in a measurable way, which is especially important in SNP Genotyping assays. Furthermore, AmpliTaq Gold DNA polymerase is more likely to displace the mismatched probe rather than cleave it to release reporter dye. More information about TaqMan MGB probes can be found in the User Bulletin entitled "Primer Express Version 1.5 and TaqMan MGB Probes for Allelic Discrimination." You can find a copy on our website by entering this title in the main search field.

Refer to the product manual for your instrument and software for specifics, but in general you will want to change the Quencher value to None.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

It is generally not possible to use DNA as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step. Therefore, in-vitro transcribed RNA is commonly used to prepare standards for the absolute quantitation of RNA targets. This would involve the cloning of the target of interest into an in-vitro transcription plasmid, performing in-vitro transcription, then purifying the resulting cRNA so that the DNA plasmid cannot serve as a PCR template. Concentration is measured by A260 and converted to the number of copies using the molecular weight of the RNA.

Relative quantitation of gene expression methods require less up-front preparation and provide a fold-change value instead of an absolute quantity result. For many researchers, absolute quantities are not a necessary parameter to measure, and therefore relative quantitation is a much more attractive approach to studying gene expression via real-time PCR. For more information on relative quantitation of gene expression, please refer to our Technical Reference Library in the Technical Resources section of our website.