Smillie and Krotkov (23) compared various methods for quantitating RNA in plant materials, and concluded that the SchmidtThannhauser procedure (20), which involves repeated tissue extractions with acid and organic solvents followed by KOH hydrolysis and use of an anion exchange resin, is the most reliable method for the widest range of plant tissues. De Deken-Grenson and de Deken (9) made the same conclusion, and described a batch method for the use of Dowex 2 in purifying the free nucleotides resulting from KOH hydrolysis of the RNA obtained by the Schmidt-Thannhauser procedure. However, the number of manipulations and the amount of sample required in the procedure described by Smillie and Krotkov (23) on the enhancement by high mol wt RNA of ethidium bromide fluorescence to estimate bound ribosomes in pea chloroplasts.Here, we describe a method for the extraction and measurement of thylakoid-associated RNA which does not require extraction or purification of undegraded RNA. Ribonucleases are added to release the thylakoid-associated RNA into the supernatant as nucleotides and oligonucleotides, thus reducing sample manipulation to a minimum. Miniature Dowex l-X2 columns are used to remove materials which interfere with analysis by UV A or the orcinol reaction for pentose.MATERIALS AND METHODS Dowex 1-X2 resin and Econo-column reservoirs were purchased from Bio-Rad. Hepes, EDTA (tetrasodium salt), pancreatic RNase T1, Torula yeast, RNA (type VI), pronase (protease type VI from Streptomyces griseus), p-aminosalicylate, and Tris base were purchased from Sigma; 8-hydroxyquinoline from Sargent-Welch; and triisopropylnaphthalene sulfonic acid sodium salt from Eastman Organic Chemicals. Pea (Pisum sativum var. Progress No. 9) seeds (captan-coated) were purchased from Agway.Columns were made by breaking off Pasteur pipettes to a final length of 10 cm, and then attaching Econo-column reservoirs at their upper ends with latex tubing and a 2.5-cm section of glass tubing. Filter paper discs (VWR No. 2831080 filter paper) cut with a No. 2 cork borer were forced to the bottom of the column with a 4-mm glass rod. A holder accommodating 30 columns were made from a 2.54-cm Plexiglas slab so that the holes for the columns were aligned with those of a test tube rack.Peas were grown for 12-16 days in Vermiculite in flats in a controlled environment chamber on a 16-h day/8-h night or a 12-h day/12-h night schedule at 21-23 C.Pea shoots were homogenized in a buffer containing 0.2 M sucrose, 200 mi Tris-HCl (pH 8.5), 60 ms KCI, and 15 mi Mgacetate. Isolation of thylakoids was performed as described by Yamamoto and Jagendorf (in preparation), and included an initial centrifugation, resuspension, and centrifuging through a washing layer of 0.5 M sucrose, collection of membranes at the lower interface following a sucrose step-gradient centrifugation (resuspended pellet without sucrose over 0.5 and 1.8 M sucrose steps), and a final washing by centrifugation. Between each step the resuspended pellets were disrupted by passing four times...