Positive control for the Cytokines assessment for allergic reactions by qPCR.
Hello Aiman Aroosh
You will have to generate positive control RNA for RT-qPCR by invitro method namely, through specific stimulation of PBMCs.
If you know which are the cytokines that are likely to be expressed in allergy reactions, then accordingly you could use a combination of stimulating agents for stimulation of the desired cytokines in PBMCs.
You may follow the protocol given in the article attached below.
Protocol in brief:
1) Isolate PBMCs by density gradient centrifugation.
2) The isolated PBMCs are cultured in vitro until maturation for 2-3 days and then exposed to different cytokine stimulators.
3) Different mitogens and antigens may be used for stimulation of the desired cytokines. You may use stimulators at the following concentrations:
Lipopolysaccharide (LPS) at 20 µg/ml,
All other stimulating agents (Peptidoglycan, PGN; Concanavalin A, ConA; phytohaemagglutinin, PHA; pokeweed mitogen, PWM) at 5 µg/ml.
4) Cells may be incubated with the stimulating agents for approximately 18 h at 37°C with 5% CO2 in 6-well plates until the expected cytokine expression reaches optimum.
5) Harvest cells and subjected them to RNA extraction using TRIzol reagent.
As per the paper attached below, for the following cytokines, the required combination of the following stimulating agents have been used.
In-vitro cytokine stimulation:
Cytokine - Stimulating agents
IL-1β - LPS
IL-4 - PWM + ConA + PHA
IL-2 - PWM + ConA + PHA
IL-6 - PWM + ConA + PHA
IL-8 - LPS
IFN-α - PGN + LPS + ConA
TNF-α - PGN + LPS + ConA
Refer to the article attached below for more information.
Article Development and Validation of a Harmonized TaqMan-Based Trip...
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