No abstract

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.

1. Tamm—Horsfall (T—H) glycoprotein has been measured by a specific radioimmunoassay method in the urine of patients with chronic renal failure, cadmium nephropathy and Lignac—Fanconi syndrome, and in renal and bladder calculi. 2. Total T—H glycoprotein excretion/24 h was markedly reduced in patients with chronic renal failure compared with normals. A highly significant correlation was observed between T—H excretion rate and creatinine clearance corrected for surface area, in both normals and patients with renal impairment. However, the mean T—H excretion per functioning nephron unit did not differ significantly in patients with renal failure compared with normals. 3. Total T—H excretion/24 h was in the normal range in patients with cadmium nephropathy despite reduced glomerular filtration rates, owing to a highly significant increase in T—H excretion per functioning nephron unit. Similar findings were obtained in a group of children with Lignac—Fanconi syndrome whose T—H excretion per functioning nephron unit was much higher than that of normal children in the same age range. This indicates an increased T—H glycoprotein excretion per functioning nephron unit in both these conditions. 4. T—H glycoprotein content of bladder and renal calculi ranged from 0002 to 5.07 mg/g of calculus. There was no correlation between T—H glycoprotein content and either the total protein content or the qualitative inorganic composition of the stones.

1.A specific and quantitative radioimmunoassay was developed for the measurement of low concentrations of human and rabbit Tamm-Horsfall glycoprotein in the presence of other proteins. Antibody-coated tubes were used as a solid phase in the assay and the optimum antibody concentration and duration of antibody coating were established.2. Pure Tam-Horsfall glycoprotein was labelled with '''1 and, because of its apparent susceptibility to radiation damage, was labelled at weekly intervals.3. Sodium dodecyl sulphate, an ionic detergent, was included in the assay at a final concentration of 0.0005% to disaggregate the glycoprotein. An overnight preincubation step in the presence of the detergent was necessary before the disaggregated glycoprotein solutions were allowed to react with the antibody. Pretreatment of the tracer with detergent was not necessary. W.2. 163Anne M. S. Grant and A. Neuberger 8. The excretion rate of Tamm-Horsfall glycoprotein in normal humans was found to be 48.1 k9.6 (SD) mg/24 h for males and 50.5+ 14.8 (SD) mg/24 h for females. The mean excretion rate of the glycoprotein in New Zealand White rabbits was 34-8 + 7.9 mg/24 h.

1. The turnover rate of urinary Tamm-Horsfall glycoprotein in rabbits was determined by two different methods. The first involved measurement of the pool size of the glycoprotein in rabbit kidney and the daily urinary excretion rate by a radioimmunoassay from which the turnover rate was calculated. 2. The second method made use of the incorporation in vivo of Na(2) (14)CO(3) and sodium [(14)C]acetate. After a single intramuscular injection of one of these compounds, urine collections were made every 24h and the glycoprotein was isolated and its specific radioactivity was determined. 3. Incorporation of the label into urinary HCO(3) (-), urea and plasma fibrinogen was also examined. The specific radio-activities of the O-acetyl, sialic acid, aspartic acid and glutamic acid residues isolated from the Tamm-Horsfall glycoprotein were compared and their half-lives were compared with that of the intact glycoprotein. The two methods gave results in quite close agreement and indicated a half-life for the glycoprotein of approx. 9h. 4. An attempt was made to localize the glycoprotein within the kidney and within the cell. It is present throughout the kidney, but was not detected in the brush-border fraction isolated from the proximal tubules. From differential cell-centrifugation studies, the glycoprotein seemed to be predominantly present in the soluble fraction (100000g supernatant). This suggests that it is either largely a soluble cytoplasmic component or is very loosely bound to a membrane, being readily released under the gentlest homogenization procedure. 5. The half-life of Tamm-Horsfall glycoprotein in human kidney was found by the radioimmunoassay method to be approx. 16h. The similarity between the composition of Tamm-Horsfall glycoprotein and human erythropoietin is discussed.