Lysate RNase protection method for soft tissues.

Estimations of RNA abundance in solid tissues by nuclease protection assays (1, 2) are difficult, because of problems attaining efficient RNA extraction and because of RNA instability during molecular hybridization or after denaturation of nuclease-treated, probe:target hybrids. A method to overcome these difficulties in the case of tissue cultured cells was published (3) which conducted molecular hybridization directly in cell lysates prepared with the strong RNase inhibitor, 5M GuSCN as described (4), then relied on sizing probe:target duplexes. Here we describe application of the method to soft tissues. Lysate RNase protection was performed with a kit from RNA LAB, Eagle PA. Lung pieces (100-300 mg) that had been incubated 24 hr in culture medium were frozen on dry ice, cut into approx 3 mmx3 mm fragments and placed in a Duall homogenizer (Kontes, Vineland NJ, cat #885451-0020). 400 /J of lysing solution (5M GuSCN/0.1 N EDTA, pH 8.0) was added for every 100 mg of tissue and the tissue was homogenized until the amber color did not increase and unhomogenized particles did not become smaller (usually 1 2 min). Homogenates were frozen at -20°C until assay. For hybridization, 20 /d of homogenate was mixed with 5 yX of P IL-la antisense or sense RNA probe and incubated at 37°C for 17 hr. RNA-RNA hybrids were digested with 10 /tg/ml of RNase A, treated with 200 /xg/ml of protease K and 1% SDS (final concentrations) and precipitated from alcohol freshly made 1 % in DEPC. RNA—RNA duplexes were analyzed by electrophoresis in 8% polyacrylaside gels. A hybridization signal corresponding to a 165 bp DNA marker was obtained both in the A1698 cells and lung homogenate (Figure 1). The signal was reduced when the hybridization step was omitted and was absent when sense probe was used. Lysates have been stable for several months at -20°C and did not show deterioration with repeated freezing and thawing. The same procedure was successful with brain, spinal cord and breast tumor biopsies. lysate