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Tech Note: Accurate Gene Expression Analysis with SYBR Premix Ex Taq II

TECH NOTE

Accurate Gene Expression Analysis with SYBR® Premix Ex Taq II

Data kindly provided by: Alyssa Sheih, graduate student at Benaroya Research Institute

Overview

In mice, exposure to cockroach allergen (CRA) provides a model for allergic airway inflammation. In this experiment, the expression of the cytokine interleukin 4 (IL-4) was measured in lung samples from mice treated with CRA. Quantitative PCR (qPCR) was performed using the SYBR Premix Ex Taq II (Tli RNaseH Plus) kit with an Applied Biosystems 7500 Real-Time PCR System. The SYBR Premix Ex Taq II kit provided accurate gene expression measurement, allowing quantification of relative IL-4 induction levels in response to CRA.

Results

The SYBR Premix Ex Taq II enzyme efficiently and specifically amplified mouse IL-4 and the GAPDH control. Relative expression of IL-4 was higher in CRA-treated lung samples than in PBS-treated samples.

Fold Expression

Relative expression levels of IL-4 as determined by qPCR. Fold expression levels of IL-4 (compared to GAPDH) in mouse lung samples from mice treated with either PBS or CRA.

Ct values for mouse <em>IL-4</em> and <em>GAPDH</em>.

Conclusion

Upregulation of the cytokine IL-4 is associated with allergies. It was expected that expression of IL-4 would increase during a CRA-induced model of allergic airway inflammation. Indeed, using SYBR Premix Ex Taq II, an increase in IL-4 relative gene expression was accurately detected in lung samples from mice that were sensitized and challenged with CRA. SYBR Premix Ex Taq II provided sensitive and accurate detection of gene expression.

Methods

Total RNA was purified from the lungs of C57BL/6 mice that were sensitized and challenged with either PBS (2 mice) or CRA (3 mice). RNA was extracted using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized from 1 µl of the extracted RNA using Superscript II RT enzyme (Invitrogen) according to the manufacturer’s protocol. Two microliters of the reverse transcription reaction (equivalent to 20 ng total RNA per 20 µl qPCR reaction) were used for qPCR with SYBR Premix Ex Taq II according to the recommended protocol. qPCR reactions were run in triplicate using probes for mouse IL-4 and GAPDH on an Applied Biosystems 7500 Real-Time PCR System. The relative gene expression level of IL-4 was determined using the delta/delta Ct method.

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