Transcriptomics for T cell differentiation

June 05, 2015

University of Nebraska Medical Center
Pharmacology and Experimental Neuroscience
Graduate Student

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Company:

Qiagen

Product Name:

RT² Profiler™ PCR Array Mouse T Helper Cell Differentiation

Catalog Number:

PAMM-503ZA-24

The aim of my research was to isolate T cells from drug-treated mice and assess genomic changes within the T cell population following treatment. I chose to use these PCR arrays because they allowed me to look at Th1/Th2/Treg/Th17 associated cytokines and transcripts for cellular differentiation. I wanted to be able to look at multiple genes associated with multiple T cell subsets, within one single sample.

Experimental Design and Results Summary

Application

RT-PCR for gene transcripts

Starting Material

cDNA converted from RNA isolated from T cells

Protocol Overview

I initially isolated mRNA from CD4+ T cells taken from drug-treated mice. I then converted the RNA into cDNA for use in the T helper cell differentiation PCR array. The 96-well plate format allows you to look at 84 different genes associated with T helper cell differentiation. It also contains all internal controls and house-keeping genes on the same plate.

Tips

High quality cDNA is needed for these plates, otherwise amplification will not occur.

Results Summary

I was able to use these RT-PCR arrays fairly easily. The primers were already in the individual wells, and I was able to make a cDNA master mix to place into each well. I achieved successful amplification of gene transcripts for each of my treatment groups. SAbioscience (Qiagen) also provides a free online analysis tool for analyzing your results easily and efficiently. Once fold changes and p values were assessed, I was able to use my results to explore different pathway analyses.

Additional Notes

In order to use the online analysis tool, you have to have at least 3 of each treatment in order to do statistics.

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Summary

The Good

Allows detection of 84 genes at one time within the same sample

The Bad

Very costly and have to use high quality DNA

The Bottom Line

The arrays allow you to look at a lot of gene changes at once, and the plates are very easy to load and use. However, they are very expensive, and the array does not come with the SYBR green needed for your PCR application. You have to buy that separately, which adds to cost.

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