Can primers for semi-quantitative RT-PCR be used for fixed-quantitative Q-PCR?

Empirical conclusion

1. If the PCR product is very short, within 200 bp, then SYBR can be used if there is no problem with Q-PCR.

2. If the PCR product is relatively long, it is best to redesign the primers.

3. If you use TaqMan for Q-PCR, you will most likely need to redesign your primers. This general product is about 100 bp, which is too large for the exo-efficiency of the enzyme. Shanghai Chuangsai Technology provides 0.5ml single-tube PCR tube, flat cover, no DNA, RNase, commodity number: C81-PCR000500-1000/pack, price 248 yuan.

4. If the efficiency of the RT-PCR primer is not good, SYBR will reduce the PCR efficiency, which will result in the PCR not being amplified. Primers can only be redesigned. Shanghai Chuangsai Technology provides 163795-75-3, SYBR Green1, fluorescent dye, BR, commodity number: D23-RS1151-100 microliters, the price is 416 yuan.

the reason

1. The shorter the product, the higher the PCR efficiency.

For qPCR, if you want to use Relative Quantification, the assumption of using the ddCT method is the efficiency of 100% PCR. Therefore, the longer the product, the more it deviates from this assumption. The less accurate the quantification. Especially when compared with the internal reference, the amplification efficiency of the internal reference is different. This produces two different regression lines. The two lines intersect because of their different efficiencies. This leads to a change in the quantitative relative relationship between the intersections, causing significant quantitative errors. This is still to be careful. Even with absolute quantification, the higher the PCR efficiency, the more sensitive the reaction and the greater dynamic range of the curve. In other words, the scope of quantification can be wider. Therefore, it generally does not exceed 200 bp. Of course, some people do make relatively large products, but generally do not recommend it.

2. The purpose of qPCR is to obtain fluorescence data instead of obtaining a PCR product.

Therefore, it is generally aimed at completing the PCR cycle as soon as possible. Naturally try to shorten the time of each cycle. If the product is too long, it will naturally take longer to extend the time. It is not necessary for qPCR.

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