2× Fast SYBR qPCR Mix

Absolute quantification and relative quantification have the following differences： 1.Absolute quantification: a method for determining the absolute quantity (copy number) of an unknown sample; It is usually used in the quantitative detection of viruses, bacteria, chlamydia, mycoplasma and the detection of genetically modified food. 2.Relative quantification: is to determine the amount of the target gene and the reference gene, and then find the relative amount of the target gene for the reference gene, and finally compare the relative amount between the samples. It was mainly used to detect the changes of mRNA expression in cells. The differences of mRNA expression in different tissues were compared. The results of gene chip and siRNA interference experiments were verified.

In order to maintain the consistency of amplification efficiency with the actual detection sample, the sample with similar structure to the actual detection sample should be selected as much as possible as the standard sample. For example, genomic DNA should be selected as the standard when genomic DNA is used as the starting material, and it is best to choose Total RNA (or cDNA synthesized with Total RNA as the template) to express the target gene as the standard when mRNA expression is analyzed. Even if the amplified base sequence is the same, but the overall template is different, it may lead to different PCR amplification efficiency (such as genomic DNA and plasmid DNA).

The first things to confirm in Real Time PCR are as follows: 1.Are there any errors in the sequences of the primers and probes? Is the combination correct？ 2.Whether Total RNA has the potential to decompose？ 3.Whether the various reagents are forgotten to add？ 4.Are the PCR conditions and fluorescence detection steps set correctly? If there is a template of successful reaction experience (PositiveControl), set up a PositiveControl reaction, it is easy to confirm the above items. The quality of Total RNA must be confirmed by electrophoresis and OD analysis。

Sometimes the Total RNA or cDNA contains substances that inhibit reverse transcription and PCR reactions (such as organic solvents used in RNA extraction). To confirm the presence or absence of such a hindrance, a higher concentration of the template was diluted in three to four gradients and used for RealTime RT-PCR reactions or RealTime PCR reactions. If there is no harmful substance, the Ct value will change depending on the template concentration. If there is a hindrance substance present, it will be found that the high concentration of the template has a decrease in the reaction performance.

When the PCR amplification efficiency calculated from the slope of the standard curve is low, the following reasons can be considered. 1.The performance of PCR was poor. The primers, reagents and PCR amplification conditions need to be explored again. 2.The incorporation of PCR blocking substances and the exploration of template extraction methods. 3.The standard dilution was not accurate. Buffer was diluted using a dedicated standard. The dilution of low concentration standards is often prone to decomposition and instability, so it is best to add tRNA and rRNA of different biological species to the diluent to play a protective role. However, there are occasional interference phenomena caused by the sequence homology of the added tRNA and rRNA with the target gene sequence.

When using chimeric fluorescence assay, nonspecific amplification sometimes leads to high PCR amplification efficiency, which needs to be confirmed by melting curve analysis.

A.The curve is smooth; the peak times is normal; there is no amplification of NTC and NRC (late peak); The Ca value is generally between 15 and 30.

For sample with Cq values of 30-35, the validity of the results can be determined by the NTC situation. Generally speaking, there is no amplification with NCT or Cq(NTC)-Cq(simple)＞5; and a Cq value greater than 35 can be considered to be no amplification.