Abstract:
Urine is an appropriate choice of specimen to study
the biomarkers for metabolic and renal disorders because it is
readily available with less harm to the patients. However, RNA
extraction from voided urine is challenging due to the presence of
RNases and cell scarcity. This study aims to optimize a protocol
for RNA extraction from urine samples for gene expression
studies in renal pathology. Hundred and two urine samples
were collected from both healthy controls (HC) (𝑛 = 15; 54 ±
11 years) and chronic kidney disease (CKD) patients (𝑛 = 87;
56 ± 10 years) and centrifuged at 6,500 ɡ for 20 min at 4 °C to
obtain sediment. RNA was extracted from urine sediments using
a phenol-based technique. The extracted RNA was quantified and
reverse-transcribed into complementary DNA (cDNA). Reverse
transcriptase quantitative polymerase chain reactions (RT- qPCR)
were carried out using 2 ng of template cDNA to amplify the
housekeeping gene, β2- microglobulin (B2M). The total yield of
RNA from CKD and HC samples were 718 ± 164 ng and 790 ±
231 ng, respectively, and a statistically significant difference was
not observed between the two study groups (p > 0.05). The urinary
RNA recovery was significantly increased with CKD progression
(p < 0.05). Further, the results show that urine volume, gender,
and serum creatinine level significantly influence the RNA yield
in only disease groups (p < 0.05). The mean threshold cycle (Ct)
values for B2M amplification of CKD and HC were 27.36 ± 3.09
and 20.97 ± 3.90, respectively. This modified phenol-chloroformbased
urinary RNA extraction method is less expensive and does
not require pre- and post-purification steps. It provides a higher
yield of RNA with less inhibition to qPCR and is sufficient for
downstream applications than column-based techniques.
