We recommend the following 3 negative controls:

-The “no template control” (NTC) is an absolute requirement for all qPCR experiments. An NTC omits DNA from the PCR. This reaction serves as a general control for unwanted nucleic acid contamination.

-A no reverse transcriptase control (–RT) omits the reverse transcriptase in the reverse transcription step of a RT-qPCR. The purpose of this control is to assess the amount of genomic DNA contamination present in an RNA preparation. Perform this control for any assay that might amplify
genomic DNA.

-A no amplification control omits the DNA polymerase from the PCR. This reaction serves as a control for background fluorescence of the PCR assay and probe stability.

For accurate analysis of qPCR results, each experiment needs to be set up with multiple controls.

While the exogenous positive controls that we offer are useful, they are intended to create calibration curves for quantitative analysis. You may not need quantified results, if only confirming a positive or negative reaction. This allows for a more cost efficient kit.

To be fully confident, it might make more sense to incorporate a know positive sample as a positive control. This will enable to verify the entire sample processing chain and avoid any mistakes.

All of our kits use the FAM reporter dye. FAM is the most popular dye, with settings for detection widely available on RT-PCR and qPCR instruments and is often more sensitive than some of the other dyes available.

The following devices are known to work with out RT-qPCR kits. Should your device not be in the list, check if it has a setting to detect FAM (518nm).

Agilent: AriaMX

Applied Biosystems: 7000, 7300 (Prism), 7500 (Prism), 7500 HT (Fast), 7700, 7900 (Prism), 7900HT, StepOne, StepOnePlus, Viia 7, QuantStudio 3, QuantStudio 5, QuantStudio 6 Flex, QuantStudio 7, QuantStudio 12K flex.

BioRad: CFX384, CFX96, Chromo4, Connect, iCycler, MiniOpticon, MiniOpticon2, MyIQ2, MyIQ5, QX100, QX200.

Cephid: Smartcycler, Smartcycler2.

Illumina: Eco.

Qiagen: Rotor-gene Q, Rotor-gene 6000

Roche: LC2.0, LC480, LC1536, LC Nano, LC96

Ensuring that qPCR is quantitative, accurate, and reproducible requires thoughtful assay design, setup, and optimization. During design, each of the MMolecular RT-qPCR assays follows stringent criteria to ensure optimal performance.

Each MMolecular assay meets the following criteria:
-PCR efficiency 2.0 +/- 0.2 (equals 100 +/- 10 %).
-Cq of highest cDNA concentration <= 34.
-Linear dynamic range of at least 3 logs.
-High amplification specificity, with no side products in gel analysis.
-Sigmoidal amplification curve.
-Fluorescence intensity of amplification curves between 5 and 50 fluorescence units.

Unopened, the dried kits will be stable for at least 3 months. We do recommend that you store our kits at -20C as soon as you receive them.

Also, it is ideal to store our RT-qPCR kits in the dark. Exposure to UV light should be avoided, and ambient lab light should be minimized.