Samples of kidney medulla and kidney cortex were collected from mice that had received i.v. injection of a) 150 MBq 177Lu-octreotate and phosphate buffered saline solution (PBS), b) 5 mg/kg A1M and PBS, or c) 150 MBq 177Lu-octreotate and 5 mg/kg A1M. Samples were also collected from control mice that had received either two injections of PBS or PBS and A1M vehicle solution. Collection of the samples was performed at the time of death; three animals in each group were killed one day after injection and another three after seven days.
Extracted molecule
total RNA
Extraction protocol
According to the manufacturer's protocol, total RNA was extracted from frozen kidney tissue samples using the RNeasy Lipid Tissue Mini Kit (QIAGEN) or AllPrep® DNA/RNA/Protein Kit (QIAGEN). The purity, integrity and concentration of isolated RNA were assessed with Nanodrop 1000 Spectrometer (Thermo Scientific), RNA 6000 Nano LabChip Kit and Agilent 2100 Bioanalyzer (both from Agilent Technologies) and Qubit 3.0 Fluorometer (Thermo Fisher Scientific), respectively. Reverse transcription was performed from 200 ng total RNA using the RT2 First Strand Kit (QIAGEN).
Label
SYBR Green
Label protocol
For each reaction, a final volume of 25 µl was used which contained 12.5 µl of RT2 SYBR Green Mastermix (QIAGEN), 11.5 µl of nuclease free water and 1 µl of cDNA (1.7 µg of total RNA). Quantitative real-time PCR were performed (7500 Fast Real-120 Time PCR system, Applied Biosystem) with thermal profile starting by activation of HotStart DNA Taq polymerase at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1min.
Hybridization protocol
n/a
Scan protocol
n/a
Description
test
Data processing
Sham-treated samples were used as a control. We compared the relative expression in kidney tissue between the controls and three treatment groups (177Lu-octreotate, 177Lu-octreotate+A1M, A1M) at one and seven days after administration. For data analysis, we used Microsoft Excel to calculate fold-expression of the target genes based upon the 2-ΔΔCt method (Livak and Schmittgen 2001). The relative expression compared to controls were analyzed by Welch T-test, and p<0.05 was considered significant. For differentially expressed genes in at least one group, one-way ANOVA followed by Welch T-test was used to determine significant differences between the 177Lu-octreotate, 177Lu-octreotate+A1M and A1M group. Target gene signals in all samples (test and controls) are normalized to geometric average of housekeeping genes provided in the same array, resulting in ΔCt values, where ΔCt=(Ct_Target-Ct_HKG). ΔCt values for all test replicates (at 1dpi and 7 dpi) were normalized against the average of corresponding control replicates, resulting in ΔΔCt, where ΔΔCt= ΔCt_test replicate– average ΔΔCt_control. 2^-ΔΔCT values for each condition were considered as fold change expression of test samples compared to controls (untreated). Fold change is determined for target genes in all replicates of test samples as 2^-ΔΔCt where -ΔΔCt= ΔCt_test replicate– average ΔΔCt_control Non-normalized raw data matrix reports Ct values for target and housekeeping genes in all control and test replicates. Normalized Ct and fold change worksheet reports ΔCt and 2^-ΔΔCt, respectivly, calculates as indicated in the data processing
