PRESERVING LYMPHOCYTE RIBONUCLEIC ACID FOR ANALYSIS AND TRANSPORT FROM WHOLE BLOOD IN RURAL AREAS.

Abstract

Background Preservation of human ribonucleic acid (RNA) from whole blood samples in rural areas with very few resources has previously been very difficult and/or expensive. This has limited clinical research trials in third world countries and distant rural areas in developed countries. The major drawback to being able to study these populations is the difficulty in transporting blood samples while maintaining the integrity and preventing degradation of the lymphocytes, DNA, protein, and RNA. Of these, RNA is the most difficult and expensive to preserve. Objective To develop an inexpensive, simple, and reliable method to collect and preserve high-quality RNA from whole blood lymphocytes for prolonged transport. Methods Blood was collected from young adult rats and treated four different ways. For the first method, blood was placed in an equal volume of RNAlater® and frozen in dry ice and then placed in −80°C for 1 week. This was then thawed on ice and processed. The second method used an equal volume of RNAlater® but was not frozen. The third method was diluted with an equal volume of water before processing. In the fourth method, fresh, undiluted blood was used. RNA was isolated with Trizol® LS immediately after collection or thawing. cDNA was then synthesized from this RNA. An RT-PCR was then run using the housekeeping gene GAPDH. Results The sample that had the highest expression was the untreated and undiluted whole blood sample at an average of 22.57 Ct. The second highest expression was the blood that was treated with RNAlater® and frozen for 1 week. This expressed at an average of 25.03 or 2.5 cycles later. The next highest expression was the blood diluted with water. The Ct value for it was an average of 30.89 or 8 cycles after the untreated blood. The fourth sample expressed at an average of 32.92 or over 10 cycles beyond the untreated blood. Conclusion We have successfully developed a simple, inexpensive, and reliable method of collection and stabilization of high-quality RNA from whole blood lymphocytes for transport and later experimental use. This preservation of RNA, when compared with fresh blood treated with RNAlater® or diluted with water, is expressed almost as highly as untreated or undiluted blood. We speculate that this procedure will greatly enhance the ability to study lymphocyte RNA in third world and rural area populations.