|
| Status |
Public on Jun 20, 2024 |
| Title |
Non-responders 4 |
| Sample type |
RNA |
| |
|
| Source name |
blood
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
|
| Treatment protocol |
Abiraterone acetate after docetaxel failure. The treatment consisted of daily oral administration of AA (1000 mg) plus prednisone 5 mg twice a day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
5-mL blood sample was obtained from each patient at the baseline (before starting AA administration). Each blood sample was collected in EDTA-treated tubes and immediately centrifuged for 15 minutes at 2500 rpm at 4°C. The plasma layer was carefully collected without disturbing the buffy coat, and stored at -80°C in 250-500 µL aliquots. Total RNA from 250 uL of plasma samples was extracted using miRNeasy® Se-rum/Plasma Kit (Qiagen) according to the manufacturer's instructions and eluted in 40 uL of DNase- and RNase-free water. Briefly, plasma samples were lysed by adding 5 volumes of QIAzol Lysis Reagent containing 1ug of MS2 phage RNA. After chloroform addition, the aqueous phase was loaded onto RNeasy Mini Spin Columns, and washing steps were performed according to manufacturer’s protocol. Qubit RNA HS Assay Kit (Thermo Fisher) was used with the Qubit Fluorometer, to provide an accurate method for the quantification of the 18 samples used for Exiqon miRNome RT-qPCR panels (Qiagen).
|
| Label |
SYBR
|
| Label protocol |
80 ng of extracted RNA were retro-transcribed using a Universal cDNA Synthesis kit (Exiqon – Qiagen) together with 4 uL of 5X Reaction buffer, 2 uL of enzyme and DNase and RNase free water up to 20 uL of total reaction volume. The reaction was performed in a T100 Thermal Cycler (BioRad) as follows: 42°C for 60 minutes and 95°C for 5 minutes. Exiqon microRNA Ready-to-Use PCR (miRCURY LNA Universal RT mi-croRNA PCR Human panel I+II, V4.M – Qiagen) was used for miRNA profiling. cDNA template was diluted 1:50 in nuclease-free water and mixed 1:1 with 2X SYBR Green Universal PCR Master Mix (Exiqon – Qiagen) for a 10 uL of total reaction volume. The reaction was performed on a CFX384 Real-Time PCR Detection System (BioRad) as fol-lows: 95°C for 10 minutes, 40 amplification cycles at 95°C for 10 seconds and 60°C for 1 minute.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Non-responder to abiraterone acetate. The patients were considered responders to AA if they either achieved a radiological response without PSA progression and performance status deterioration, or in the presence of PSA reduction ≥ 50% compared to the baseline values without radiological progression and performance status deterioration
|
| Data processing |
Matrix non-normalized represents non-normalized Ct values. miRNAs non-detected in at least 20% of the samples were removed and Ct equal or higher than 40 were considered non-detected. Matrix normalized template represents Ct values of panels normalised on miR-425-5p. Fold change template represents responders (R) evaluated relative to non-responders (NR) [2^-(ΔCt NR – ΔCt R)]
|
| |
|
| Submission date |
Mar 26, 2024 |
| Last update date |
Jun 20, 2024 |
| Contact name |
Simone Detassis |
| E-mail(s) |
simone.detassis@unitn.it
|
| Organization name |
University of Trento
|
| Department |
CIBIO
|
| Street address |
Via Sommarive 9
|
| City |
Trento |
| ZIP/Postal code |
38123 |
| Country |
Italy |
| |
|
| Platform ID |
GPL34334 |
| Series (1) |
| GSE262550 |
qPCR panels for plasma microRNAs of responders versus non responders metastatic castration resistant prostate cancer patients to abiraterone acetate |
|
