The study was conducted at Sri Ramachandra institute of Higher Education and Research form the Department of Nephrology SRIHER, Chennai ,India .The children who have met the criteria’s have been rolled for the study with the age group from 1–12 years. For this study the sample size was taken as 300 among which 200 cases (SSNS,SRNS) and 100 aged matched control groups.
The inclusion criteria was involved as the histological confirmation with or without the familial history of NS along with the histological confirmation of MCNS or FSGS. Then the children who do not respond to prednisilone therapy which requires minimum period of 4 weeks has been included. Secondary NS and SRNS with the history other than FSGS or MCNS are taken as exclusion criteria. In the study proper inform consent from all the parents/guardian along with the asset form was obtained. Ethics committee approval has been obtained from the Institutional Ethics committee at Sri Ramachandra Institute of Higher Education and Research [IEC-NI/21/FEB/77/28]
2.3 Sample collection
About 6 ml of urine that was taken in a sterile container from children aged 1 to 12 years. A total of 200 cases of SSNS and SRNS, along with 100 children who were age-matched controls, As soon as the samples were obtained, they were processed by centrifuging them for 20 minutes at 3,000 rpm, discarding the supernatant.2.4 Isolation of RNA from the urine pellet.
The urinary pellet was lysed with RNA lysis buffer and it was stored in the deep freezer at 80°C for further purpose. After that MicroRNA isolation was carried out for both the cases SSNS ,SRNS and for the control groups by using the MiRNeasy Mini Kit and the procedure was performed according to the manufactures protocol. Urine samples were mixed thoroughly with an equal volume of miRNeasy Serum/Plasma Spike-In Control, incubated for 2–3 minutes at room temperature, and then analysed.centrifuged at 12,000 g for 15 minutes at 4°C. After transferring the upper aqueous phase to a new collecting tube, 1.5 litres of 100% ethanol were added. A 2 ml collection tube containing 700 l of material was pipetted into an RNeasy Min Elute spin column and spun at 8000 g for 15 s at room temperature. The supernatant was thrown away. The RNeasy Min Elute spin column received about 500 l of elution buffer, and it was centrifuged at the same speed. The RNeasy MinElute spin column was filled with approximately 500 l of 80% ethanol and centrifuged. The concentrations and purity of the samples were calculated from the isolated nucleic acids. On the NanoDrop 1000, the concentration and quality of the isolated RNA were evaluated using spectrophotometry (Thermo Scientific, Waltham, MA). To determine how pure a nucleic acid is, utilise the A260/280 ratio, which is the absorbance at 260 and 280 nm. The lower pedestal of the nano drop was filled with around 2 l of the separated sample, and using the nano drop apparatus, the nucleic acid purity of each collected urine sample was determined. Most people considered a ratio of 1.7–2.0 to be of good quality2.6 All the 300 samples were checked for quality and quantity are found to be good and with adequate proportion for expression study40.
2.5 Gene Expression Analysis using quantitative real time PCR
The cDNA conversion was performed using the Taqman advanced cDNA synthesis kit and the converted cDNA templates were further diluted to proceed for microRNA expression analysis using Rotar Gene Q software for the selected miRNAs miR-17-5P,miR-155p ,miR- 424 -5p ,miR-1 and 215.The expression levels for all the microRNAs was normalized using miR- 484-5p as endogenous control.
2.7 Computational prediction for microRNAs using online data base
MicroRNA prediction tool such as miRWalk and miR Target link human (sticht C,De Torre C,Parveen A,Gretz et al 2018) which was useful to analyse and the select the specific target MicroRNAs with the database available for the study and experimentally validated predicted microRNAs were further analysed using the g profiler software in order to identify and the select the specific function relate to this disease.