Modified tissue pulverization technique and evaluation of dihydrofolate reductase amplification as a pan-tissue RT PCR control.

Department of Medicine, Section of Rheumatology, University of Minnesota, Minneapolis, Minnesota 55455 When processing large numbers of tissue samples for amplification by reverse transcriptase polymerase chain reaction (RT PCR), certain factors must be considered. Reuse of tissue homogenizat ion equipment and handling of multiple samples increase the probability of contamination, leading to false-positive reactions. Additionally, studies that examine a variety of tissues or cell types require a universally expressed gene as an amplification control. These concerns were initially addressed as a result of our attempts to amplify extremely low levels of persistent coxsackievirus RNA from skeletal muscle. Isolation of intact RNA from skeletal muscle is difficult because of the small amount of tissue available and the comparatively low levels of RNA. These drawbacks are compounded by the presence of connective tissue, which makes tissue homogenizat ion difficult. To overcome these difficulties, we developed a modification of a previously reported method (1~ that requires little equipment, minimizes the potential for contaminat ion, and facilitates the isolation of high-quality RNA from frozen mouse muscle. It has also proved effective for pulverization of a wide variety of tissues. We then used this RNA to test the feasibility of using dihydrofolate reductase (DHFR) as a pan-tissue amplification control. Mouse tissue was harvested, placed in cryotubes, flashfrozen in liquid nitrogen, and stored at 70°C. During pulverization, all tissue samples were held on dry ice. To prevent cross-contamination, a separate nylon forceps was used to handle each tissue sample. Before reuse, forceps were washed, soaked in 0.5 N NaOH to hydrolyze contaminat ing RNA, rinsed in water, and autoclaved. For each sample, an envelope of a luminum foil was prepared, which was four layers thick and at least five times larger than the tissue sample. Just before use, the envelope was dipped in liquid nitrogen and the tissue was placed inside. This was dipped in liquid nitrogen again and placed on a foil-covered 2.5-cm-thick iron plate stored at -70°C and posit ioned on a bed of dry ice. The tissue was completely pulverized by striking it several times with a hammer . The pulverized tissue formed a disk that could be picked up with forceps after flexing the foil and transferred to a 1.5-ml microcentrifuge tube containing 520 txl of solution D. (z~ The tissue was immediately vortexed on medium-high speed for 10-20 sec, and then microcentrifuged for 10 sec to pellet insoluble debris. A 500 btl volume was transferred to a fresh tube, leaving behind tissue debris that might trap RNases. (3) At this point the sample was kept at room temperature (for a m a x i m u m of 30 min) until remaining samples were processed. RNA isolation was cont inued as described. (2) RT PCR amplification of DHFR employed a 25-mer sense primer DHFR-S2 (5'-TCG ACC ATT GAA CTG CAT CGT CGC C-3') from exon I and a 26-mer antisense primer DHFR-A1 (5'-GGA ATG GAG AAC CAG GTT TTC CTA CC-3') from exon III, based on the published sequence for mouse DHFR. (4) These primers span nucleotides 6-185 of the mRNA. Reverse transcription of DHFR was performed in a 20-b~l reaction containing 10 pmoles of DHFR-A1, 20 units of RNasin, and 50 units of Moloney murine leukemia virus (MMLV) RT (GIBCOBRL, Bethesda, MD), with buffer components as r ecommended by the manufacturer. The reaction was incubated at 37°C for 1 hr, 95°C for 5 min, and cooled to 5°C. PCR was performed by adding 10 pmoles of DHFR-S2 and additional reagents to obtain final reaction conditions of 1.5 mM MgC12, 40 mM KC1, 10 mM Tris (pH 8.3), and 2.5 units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN) in a final volume of 100 ILl. Thirty-five cycles of 94°C for 1 min, 50°C for 1.5 min, and 72°C for 1.5 min were followed by a final extension at 72°C for 8 min. On the average, we recovered 26 ~g of RNA from 30 mg wet weight of mouse hindquarter skeletal muscle, with an ODz6o/28 o ratio of 1.8. This is comparable to reported muscle RNA yields. (1) P.NA was obtained from samples of mouse muscle weighing up to 100 mg without increased degradation or loss. Densitometric tracings of rRNA bands yielded an average 285/185 ratio of 1.8 (s) The quality of RNA extracted from mouse spleen, liver, kidney, diaphragm, thymus, heart, brain, spinal cord, and small intestine was comparable to that of mouse skeletal muscle. One exception was the pancreas, which had a 28s/18s ratio of 1.1 and is known to possess high levels of RNase. Disruption of muscle by other methods such as vortexing with glass beads or hand-held homogenizers yielded similar recoveries of RNA but of less consistent quality. Aerosolization was also more