Oct 07, 2022
Real-time quantitative polymerase chain reaction (RT-qPCR)
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Protocol Citation: An.Huang, Qiaowa Gong 2022. Real-time quantitative polymerase chain reaction (RT-qPCR). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkkqmxl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 20, 2022
Last Modified: October 07, 2022
Protocol Integer ID: 67101
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Abstract
Real-time quantitative polymerase chain reaction (RT-qPCR) helps determine the expression level of a certain gene by amplifying DNA according to the target mRNA template. This protocol describes the procedure of conducting RT-qPCR using Promega GoTaq® qPCR Master Mix A6001.
Materials
8-Tube Strip 0.1 ml, sterile, aerosol-resistant pipette tips, nuclease-free pipettors, cDNA template and qPCR primers (reference gene and target gene), GoTaq® qPCR Master Mix.
Protocol materials
GoTaq(R) qPCR Master Mix, 40 reactionsPromegaCatalog #A6000
Step 1
Thaw the GoTaq® Master Mix and Nuclease-Free Water. Thaw the cDNA templates and primer. GoTaq(R) qPCR Master Mix, 40 reactionsPromegaCatalog #A6000
Note
Do not thaw the GoTaq® Master Mix at temperatures above room temperature.
Vortex the GoTaq® Master Mix for 3–5 seconds to mix. Vortex at low speed to avoid aeration.
Determine the number of reactions to be set up, including negative control reactions. Add 1 or 2 reactions to this number to compensate for pipetting error.
Note
While this approach does require using a small amount of extra reagent, it ensures that you will have enough reaction mix for all samples.
Prepare the reaction mix (minus DNA template) by combining the GoTaq® qPCR Master Mix, PCR primers and Nuclease-Free Water as described below. The DNA template is added in Step 6. Vortex briefly to mix.
The component of reaction mix. Calculate the volume of primer according to the concentration of primer used.
Add the DNA template (or water for the no-template control reactions) to the appropriate wells of the reaction plate.
Seal the tubes or optical plate, and centrifuge briefly to collect the contents of the wells at the bottom. The samples are ready for thermalcycling.
Note
Protect the samples from extra light exposure or elevated temperatures.
Thermalcycle in qPCR equipment. Realtime qPCR is performed with an initial denaturation of 3 min at 95°C, followed by 40 cycles of 20 s at 95°C, 20 s at 60°C, and 20s at 72°C.