Phenol based rna isolation is the preferred procedure for study of gene expression in human urinary sediment
1. 3. Urology-Nephrology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Evaluating the expression of genes in urinary sediment has been considered as a promising non-invasive approach for discovery of biomarkers of renal diseases. nonetheless, extracting rna from this valuable source of biomarkers is notoriously challenging due to low cellular content and other factors that affect the quality of isolated rna. hence, we compared four different methods for isolation of rna from urine sediment samples. trizol reagent with basic protocol (method 1), modified procedure of trizol (method 2), a column-based protocol (method 3) and combination of method 1 and 3 (method 4) were applied for isolation of rna from identical aliquots of five healthy urine samples. concentration and purity of isolated rnas were assessed and cdna synthesis was performed. expression level of gapdh and mir-21 was studied by quantitative rt-pcr. the highest yield of rna extraction was observed in method 1 and 2. no difference in purity of rna in different methods was noticed. quantitative rt-pcr findings revealed the lowest levels of ct values (higher expression) in samples of method 1. although concentrated rnas were isolated from samples of method 2, the declined ct values could indicate degradation of isolated rna. column based protocols (method 3 and 4) were not capable of significant recovery of rna. trizol isolation, as a phenol based method, is the most straightforward and reliable procedure for rna isolation from urinary sediment cells.
We used 4 different isolation methods for each sample and compared rna yield and purity. (i) method 1, rna isolation was performed by trizol reagent (thermo scientific, waltham, ma, usa) according to manufacturer instruction and rna pellet was dissolved in 30 μl of diethyl pyrocarbonate (depc) water. (ii) method 2 was based on trizol reagent with some modifications. we have previously used this method for isolation of total rna enriched in mirnas . in this method the same procedure was used for isolation of aqueous phase (containing rna fraction). afterward, 1 ml of 100% ethanol was used instead of isopropanol and the mixture was incubated at -20 ºc for overnight. rna pellet was precipitated by centrifugation for 45 min at 14000 ×g in 4 ºc. pellet was washed with 75% ethanol and centrifuged for 8 min at 14000 ×g in 4 ºc. to facilitate rna precipitation, 0.35 of glycogen (20mg/ml) was added to aqueous phase in method 1 and 2. (iii) method 3, a column based technique was performed by using fastpure rna kit (takara) per manufacturer’s instruction. (iv) method 4 was an integration of method 1 and method 3. briefly, lysis of sample was carried out by trizol reagent and aqueous phase was separated according to method . then, aqueous phase was mixed with 500 μl of 70% ethanol and the procedure was followed according to fastpure rna kit (takara) instruction. the rna concentration was measured by absorbance at 260 nm and the purity of rna was assessed by the ratio of absorbance at 260 to 280 nm using wpa spectrophotometer (biochrom).
Rna concentrations in method 1 (219 ± 62.8 ng/µl) and 2 (235.6 ± 42.6 ng/µl) were significantly higher than method 3 and 4 . no significant differences in 260/280 and 260/230 ratio were observed between different isolation methods . rt-pcr of gapdh as a housekeeping gene showed positive and specific amplification in all 5 samples (a-e) of method 1 but only in one of the samples of methods 2 and 3, specific pcr products were observed. results of gapdh expression may be indicative of extensive rna degradation in method 2-4. it has been revealed that mirnas are relatively stable rna species of urine . so, we evaluated the expression of mir-21 in our samples. contrary to the expression of gapdh, mir-21 has been detected in most of samples . intriguingly, mir-21 has been detected in significantly higher levels in rna samples processed in method 1.
Evaluation of gene expression levels in urinary sediment would be an ideal non-invasive surrogate marker for different renal diseases . measurement of mrna expression level could be considered as better experimental tool in comparison with protein assessment especially for renal diseases as it is not affected by glomerular filtration and tubular reabsorption .
Rna isolation, urinary sediment, quantitative rt-pcr.