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Ribonuclease Protection Assay System Offered by PharMingen Human Apoptosis Multi-Probe Template Set. Autoradiograms of protection assays using human APO-2 template set and various cell lines. Quantitating expression levels, especially of rare mRNA species, has never been an easy task. Only a few options exist for doing this, and each has difficulties. The ribonuclease (RNase) protection assay, a sensitive and specific method for quantitating expression levels, has been around for close to 15 years (D. Zinn et al., Cell, 34:865-79, 1983) with only minor improvements. Now PharMingen has a set of reagents, Multi-Probe Template Sets to be used with its Riboquant® Ribonuclease Protection Assay System, designed to improve the sensitivity of detection and to simplify quantitation of mRNA levels. These kits contain collections of probes for related mRNA molecules--as many as 12 per set--which enable simultaneous measurements of all the messages in the set. RNase protection assays work in the following way: radioactive RNA probes are made by transcribing cloned DNA templates with one of the several available RNA polymerases (T7, in the case of the PharMingen kit). These labeled probes are then hybridized in solution to either purified mRNAs or total RNA. After hybridization, residual probe, as well as unhybridized regions of mRNA, is removed by digestion with a mixture of RNases, and the protected probe (labeled probe/mRNA hybrid) is run out on gels. PharMingen's Multi-Probe sets combine 8 to 12 templates of related molecules in a single reaction mix (a microliter of reaction mix contains 50 ng of each template). When working with precious samples or with mRNAs expressed at low levels, these template sets allow for multiple determinations to be made from a single sample. The probe sets are designed such that each gives a distinct size fragment after hybridization to target mRNA, enabling the simultaneous detection of all members of the cohort. And finally, included in the set are internal standards consisting of two constitutively ex-pressed housekeeping genes, which can be used to normalize the data from different samples and even from different experiments. Howard Young, Head of the Cellular and Molecular Immunology Section at Frederick Cancer Research and Development Center, Frederick, Md., has used Pharmingen's kit, has written about it, has talked about it, and has even worked with the company to improve it. According to Dr. Young, the major advantage of the kit lies in its ability to analyze many genes at once, particularly when material is limited. While this kit competes directly with DNA chips, chip technology at present is well beyond the reach of most research budgets. In press are several studies of Young's using PharMingen probe sets to analyze expression of apoptosis genes in tumor cells and cytokine genes in subpopulations of lymphocytes. With the sensitivity provided by this kit, Dr. Young has discovered some unexpected gene expression in these studies. While some of the first probe sets focused on genes of the immune system--cytokines, chemokines, cytokine and chemokine receptors--PharMingen is expanding into other areas of investigation, with template sets for cell cycle regulators, apoptosis-related proteins, and proto-oncogenes. The system is comprised of Multi-Probe Template Sets (sufficient for 10 transcription reactions), an In Vitro Transcription Kit (for 25 reactions), and an RNase Protection Assay Kit for analyzing 200 individual samples, which may be purchased together or separately. For more information, call PharMingen at 800-825-5832 or visit the company web site at www.pharmingen.com.

Measure Up To 12 Different mRNAs In A Single Sample With Riboquant(R) Multi-Probe Ribonuclease Protection Assay System Offered by PharMingen

The Scientist Staff

Mar 29, 1998

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