EVALUATION OF 3 RAPID RNA EXTRACTION REAGENTS - RELEVANCE FOR USE IN RT-PCR AND MEASUREMENT OF LOW-LEVEL GENE-EXPRESSION IN CLINICAL-SAMPLES

Although peripheral blood and bone marrow are usually readily availabl e from patients, present techniques of RNA extraction are tedious, req uire millilitres of starting material and removal of red blood cells b efore RNA purification. Further, successful reverse transcriptase poly merase chain reaction (RT-PCR) amplification requires the removal of h aemoglobin derivatives which interfere with the PCR process. Recently, one step rapid use reagents have become available, claiming to be use ful for obtaining high quality RNA from microlitre quantities of whole blood drawn directly from the patient. Their use to date in clinical samples appears limited with little information in the literature docu mented. In an attempt to overcome this, we tested the Trizol-LS(R), RN A-STAT-50(R) and Ultraspec-3(R) reagents upon a statistically signific ant number of clinical isolates of fresh and cryopreserved peripheral blood, bone marrow, blood apheresis products and a breast cancer cell line (MCF7) in order to evaluate whether these methods could be applie d to routine laboratory use in an RT-PCR method capable of detecting r are gene expression. Our findings showed that there was some variation in the quality of RNA extracted which was indicated by absorbance spe ctrophotometry at 260 and 280 nm. 1% agarose gel electrophoresis showe d that each of these methods could yield total RNA capable of generati ng the signature 18S and 28S rRNA bands. Using the Kruskal-Wallis non- parametric anova test combined with Dunn's multiple comparison test, t he only statistically significant difference (p<0.05) indicated that T rizol-LS(R) was more reliable than RNA-STAT-50-LS(R) and Ultraspec-3(R ) at extracting RNA from fresh peripheral blood. RNA extracted with th e Trizol-LS(R) and RNA STAT-50(R) reagents was successfully amplified in a multiplex RT-PCR reaction for detection of the multi-drug resista nce related genes MDR1, the multi-drug resistance related protein (MRP ) and topoisomerase II alpha. Low level MDR1 gene expression could be detected in frozen whole blood. However, PCR products were only seen w hen the anti-coagulant heparin was removed from all samples prior to c DNA production. RT-PCR amplification was not 100% successful with RNA extracted with Ultraspec-3(R) reagent. In conclusion, we found that th e RNA extracted from whole blood with the Trizol-LS(R) and the RNA-STA T-50(R) are suitable for use in clinically relevant molecular biology protocols that analyze rare event genes without further purification. Our results indicated that the Trizol-LS(R) reagent was generally more consistent in obtaining a pure and sufficient quantity of RNA from pa tient material as shown by the mean result of purity and quantity in c omparison to either Ultraspec-3(R) or RNA-STAT-50-LS(R) reagents. Ultr aspec-3(R) is not easily suited for direct use with whole blood produc ts.