RT-PCR of microRNAs derived from rats: Ischaemia, Sevoflurane preconditioning, Ischaemic preconditioning, Sham model
We compared sevoflurane preconditioning and ischaemic preconditioning effects against renal ischaemia reperfusion injury via microRNAs of the kidney that promote cell-survival pathways in rats. In our protocol, male Wistar rats were divided into four groups (Ischaemia, Sevoflurane preconditioning, Ischaemic preconditioning and Sham models; n=7 each) and all underwent right nephrectomy. Ischaemia model rats underwent 45-minute clamping of left kidney, followed by 4-hour reperfusion. Sevoflurane preconditioning involved exposure to 1 MAC sevoflurane for 15 minutes. Ischaemic preconditioning included 3 cycles of 2-minute clamping and 5-minute reperfusion. Renal biopsy samples were assessed postoperatively to comprehensively analyse renal mircoRNAs using RT-PCR and the predicted cell-survival pathways. Sevoflurane preconditioning and ischaemic preconditiong ameliorated ischaemia reperfusion injury, indicated by serum creatinine equally. The result of RT-PCR indicated that sevoflurane and ischaemic preconditioning affected different kinds of microRNAs related to the cell-survival pathways respectively.
Steps to reproduce
All the 28 kidneys sampled were analysed. The left kidney of the the rat used was sampled and permeated with RNA later solution (Applied Biosystems, Foster City, CA, USA) and stored at -80℃. After thawing, the kidney was sufficiently crushed in liquid nitrogen, and total RNA was extracted using the mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) and assessed with the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, the rat renal cyclic DNA was reverse transcribed and the specific miRs were amplified and measured with TaqMan Low Density Array (Applied Biosystems, Foster City, CA, USA) containing 398 kinds of well-known miRs derived from rodents in the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using a comparative quantification method. The results of RT-PCR were analysed with Data Assist software (Applied Biosystems, Foster City, CA, USA) as follows: ・ The control gene was set as Y1, which has the highest stable value among the candidate. ・ The ΔCt value of each miR was calculated by comparing the number of amplifications until it exceeded the automatic threshold value with that of the control gene Y1. ・ The ΔΔCt value was obtained by subtracting the mean ΔCt value of the Sham group from that of each group, and miR expression value, as the Fold Change (F.C. = 2 to the -ΔΔCt power), was obtained. ・ The adjusted F.C. (adjF.C.) value of each miR in each group was calculated by dividing its F.C. value by the mean F.C. value of the Sham group All values were normally distributed and inter-group comparisons were made using Tukey’s HSD test. One-way analysis of variance (ANOVA) was performed for miR in which the adjF.C. in APC or IPC satisfied the following conditions: 1. AdjF.C. ≥1.2, and not less than double the IRI group adjF.C. 2. AdjF.C. ≤0.8, and not more than half the IRI group adjF.C. Furthermore, multiple comparisons by Tukey's HSD test were performed on the miRs having significant inter-group differences (p<0.05) in mean adjF.C. values. R was used in all statistical tests. (R is a language and environment for statistical computing and graphics similar to S [http://cran.r-project.org/]).