miRNAs were extracted from baseline plasma using the Qiagen miRNeasy Serum/Plasma miRNA extraction kit, using adaptations previously described [23] (link). Briefly, 250 µl of plasma was centrifuged at 3000×g for 7.5 minutes at room temperature (RT). Then, 200 µl of supernatant plasma was transferred to a PhaseLock Gel 15 ml tube (5 Prime Inc.) containing 2.0 mL Qiazol (Qiagen), inverted 10 times, and incubated for 5 minutes at RT. After denaturation (but prior to extraction, described below), each sample received a well-mixed, single-use aliquot containing 30 fmol of synthetic C. elegans miRNA (cel-miR-39; Integrated DNA Technologies) and 1 µg of carrier rRNA (Roche Life Sciences, cat# 10 206 938 001) in Qiazol. The sample was inverted 10 times, chloroform (0.2x volume) was added and the sample was inverted again 10 times, and incubated at RT for 2 minutes. The samples were centrifuged at 1500×g for 5 minutes at RT and processed following the remainder of the miRNeasy extraction kit protocol.
Qiazol
Manufactured by Roche
Sourced in Germany
Qiazol is a reagent used for the isolation of total RNA from cells and tissues. It is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components. Qiazol efficiently disrupts cells and denatures proteins while maintaining the integrity of the RNA.
Automatically generated - may contain errors
4 protocols using qiazol
1
Plasma miRNA Extraction and Normalization
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated using Qiazol (Roche, Berlin, Germany) as per the manufacturer’s instructions. cDNA was synthesized from 5 µg total RNA and used for quantitative real time reverse transcription polymerase chain reaction (RT PCR) analysis. RT PCR was carried out under the following conditions: 95 °C for 3 min (pre-incubation), followed by 40 cycles of 95 °C for 20 s, 62 °C for 20 s, and 72 °C for 20 s. The comparative critical threshold (Ct) method was used for the calculation of expression fold change between samples. Gene expression was calculated as fold induction, relative to the mean value of control samples using the equation 2−∆∆Ct. GAPDH was used as the housekeeping gene. Primers used are shown in Supplementary Table S1 . Experiments were performed in triplicate.
3
Plasma microRNA Extraction for Biomarker Discovery
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MicroRNAs were extracted from baseline plasma (prior to surgery) using the Qiagen miRNeasy Serum/Plasma miRNA extraction kit, following modifications as previously described (Ng et al, 2009 (link)). Briefly, 250 μl of blood sample was centrifuged at 3000 g for 7.5 min at room temperature (RT). Then, 200 μl of supernatant plasma was transferred to a PhaseLock Gel 15 ml tube (5 Prime Inc.) containing 2.0 ml Qiazol (Qiagen, Hilden, Germany), inverted 10 times, and incubated for 5 min at RT. After the denaturation step and prior to extraction (described below), each sample received a well-mixed, single-use aliquot of 30 fmol of synthetic C. elegans miRNA (cel-miR-39; Integrated DNA Technologies, Coralville, IA, USA) and 1 μg of carrier rRNA (Roche Life Sciences, Penzberg, Germany, cat# 10 206 938 001) in Qiazol. The sample was inverted 10 times, chloroform (0.2 × volume) was added and the sample was inverted again 10 times, and incubated at RT for 2 min. The samples were centrifuged at 1500 g for 5 min at RT and processed following the relevant portions of miRNeasy extraction kit protocol.
4
Immunoaffinity Enrichment of AGO2 Protein and miRNAs
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A 200 μL aliquot of Magna Bind goat anti-mouse IgG Magnetic Bead (Thermo Fisher Scientific, Waltham, MA, USA) was washed 3 times with PBS solution (300 μL), and incubated with 10 μg of mouse monoclonal anti-AGO2 (ab57113; Abcam, Cambridge, UK) or mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies for 2 h at 4 °C. The preincubated beads and antibody were then added to 400 μL of urine and incubated overnight at 4 °C. Beads were washed 3 times with 1% Nonidet P-40 buffer (1% Nonidet P-40, 50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 2 mM EDTA) and resuspended in 200 μL of PBS. One half of each of 10 control samples was eluted in loading buffer, following which 5 samples were analysed by SDS/PAGE and immunoblotting with rabbit polyclonal anti-AGO2 antibody (ab32381; Abcam) with a positive control extracted from human embryonic kidney (HEK)293 cells. The other half was eluted in 750 μL of QIAzol plus carrier RNA (MS2 RNA, Roche) and processed for RNA isolation and miRNA detection.
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