The miRNeasy Mini Kit (QIAGEN, Germany) was applied to isolate total RNA, including small RNAs, from plasma. qPCR analysis was performed as described previously (Mussbacher et al., 2020 (link)). Robustness of RNA extraction, complementary DNA synthesis, and qPCR amplification was assessed using combinations of synthetic spike-in controls (Exiqon, Denmark). Hemolysis was assessed using the ratios of miRNA-23a-3p and miRNA-451a and positive samples were excluded from the analysis.
Synthetic spike in controls
Manufactured by Qiagen
Sourced in Denmark, Germany
Synthetic spike-in controls are laboratory reagents designed to monitor the performance of nucleic acid extraction, amplification, and detection workflows. They are typically made of synthetic DNA or RNA sequences that are added to samples during processing to assess the efficiency and consistency of the analytical process.
Automatically generated - may contain errors
8 protocols using synthetic spike in controls
1
Plasma miRNA Isolation and Quantification
2
Automated Plasma miRNA Extraction
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Total RNAs including miRNAs were extracted from exactly 200 μl of EDTA plasma samples using an automated Maxwell system (Promega, WI, USA) with the respective miRNA isolation kit (miRNA tissue lysis kit, Promega) according to the manufacturer’s instructions. A mix of three synthetic spike-in controls (Exiqon, Denmark) was added to the lysis buffer prior to isolation in order to monitor RNA extraction efficiencies.
3
Serum Total RNA Extraction Protocol
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Total RNA was extracted from 100 μL of serum using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) as described by Kocijan et al. [25 (link)]. Then, 100 μL of each serum sample was mixed with 1000 μL of Qiazol and 1 μL of a mix of 3 synthetic spike-in controls (Exiqon, Copenhagen, Denmark). After a 10-min incubation at room temperature, 200 μL of chloroform was added to the lysates followed by centrifugation at 12,000× g for 15 min at 4 °C. Then, 650 μL of the upper aqueous phase was transferred to a miR-Neasy mini column, where RNA was precipitated with 750 μL of ethanol followed by automated washing with RPE and RWT buffer in a QiaCube liquid handling robot. Finally, total RNA was eluted in 30 μL of nuclease-free water and stored at −80 °C.
4
Serum RNA Extraction for Profiling
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Total RNA was extracted from sera using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Samples were thawed on ice and centrifuged at 12.000 rpm for 5 min to remove any remaining cellular debris. For each sample, 200 µl of serum was mixed with 1,000 µl Qiazol and 1 µl of a mix of three synthetic spike‐in controls (Exiqon, Vedbaek, Denmark). After 10 min incubation at RT, 200 µl chloroform was added to the lysates followed by centrifugation at 12,000 rpm for 15 min at +4°C. Precisely 650 µl of the upper aqueous phase was mixed with 7 µl glycogen (50 mg/ml) to enhance precipitation. Samples were then transferred to a miRNeasy mini‐column, and RNA was precipitated with 750 µl ethanol followed by automated washing with RPE and RWT buffer in a QiaCube liquid handling robot. Finally, total RNA was eluted in 30 µl nuclease‐free water and stored at −80°C until further analysis.
5
Robust qPCR for miRNA Analysis
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qPCR analyses were performed as described.17 Robustness of RNA extraction, complementary DNA (cDNA) synthesis, and qPCR amplification was assessed using combinations of synthetic spike‐in controls (Exiqon, Denmark). Hemolysis was assessed using the ratios of miRNA‐23a‐3p and miRNA‐451a.18 Of note, no samples failed because of hemolysis or high analytical variance.
6
Serum microRNA Isolation Protocol
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Serum samples were thawed at room temperature followed by centrifugation at 12,000×g for 5 min at 4 °C to remove any cellular debris. Extraction was conducted by use of the miRNA easy QIAgen kit. For homogenization, 200 µl of serum were mixed with 1000 µl Qiazol and 1 µl of a mix of three synthetic spike-in controls (Qiagen, Germany). After a 10-min incubation at room temperature, 200 µl chloroform were added to the lysates followed by cooled centrifugation at 12,000×g for 15 min at 4 °C. Precisely 650 µl of the upper aqueous phase were mixed with 7 µl glycogen (50 mg/ml) to enhance precipitation. Samples were transferred to a miRNeasy mini column, and RNA was precipitated with 750 µl ethanol followed by automated washing with RPE and RWT buffer in a QiaCube liquid handling robot. Finally, total RNA was eluted in 30 µl nuclease free water and stored at −80 °C until further analysis.
7
Extraction of Serum RNA Using miRNeasy Kit
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Total RNA was extracted from 200 μL serum using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Samples were thawed on ice and centrifuged at 12,000g for 5 minutes to remove any cellular debris. For each sample, 200 μL of serum were mixed with 1000 μL Qiazol and 1 μL of a mix of three synthetic spike‐in controls (Qiagen). After a 10‐minute incubation at room temperature, 200 μL chloroform were added to the lysates followed by cooled centrifugation at 12,000g for 15 minutes at 4°C. Precisely 650 μL of the upper aqueous phase were mixed with 7 μL glycogen (50 mg/mL) to enhance precipitation. Samples were transferred to a miRNeasy mini column where RNA was precipitated with 750 μL ethanol followed by automated washing with RPE and RWT buffer in a QiaCube liquid handling robot. Finally, total RNA was eluted in 30 μL nuclease‐free water and stored at −80°C to await further analysis.
8
Serum RNA Extraction Protocol
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Total RNA was extracted from 100 µL serum using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Samples were thawed on ice and centrifuged at 12,000× g for 5 min to remove any cellular debris. For each sample, 200 µL of serum was mixed with 1000 µL Qiazol and 1 µL of a mix of 3 synthetic spike-in controls (Qiagen, Hilden, Germany). After a 10-min incubation at room temperature, 200 µL of chloroform was added to the lysates, followed by cooled centrifugation at 12,000× g for 15 min at 4 °C. Precisely 650 µL of the upper aqueous phase was mixed with 7 µL glycogen (50 mg/mL) to enhance precipitation. Samples were transferred to a miRNeasy mini-column where RNA was precipitated with 750 µL ethanol followed by automated washing with RPE and RWT buffer in a QiaCube liquid handling robot. Finally, total RNA was eluted in 30 µL of nuclease-free water and stored at −80 °C to await further analysis.
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