Before surgical tumor resection, blood samples were collected in clot activator tube from each participant. The basic demographic and clinical information details were taken from all participants. Depending upon histological type and grade of tumor and cancer stage, staging of tumors have been done according to American Joint Committee on Cancer (AJCC) (Mitchell et al., 2008; Schwarzenbach et al., 2011). For serum separation, blood was kept for 45 minutes to allow clotting and thereafter it was processed according to Qiagen Kit (Germany) protocol. The Blood was centrifuged at 40C for 3,000 rpm for 10 minutes (REMI). To remove other contaminants like erythrocytes the supernatant fluid was re-centrifuged at 4°C at 13,500 rpm for 10 mins to yield better utility of miR for further processing of total RNA isolation. The Serum was stored at -80°C until processing for total RNA isolation. According to manufacturers protocol, total RNA were extracted from serum samples using Qiazol reagent (Qiagen, Germany). For this, the miRNeasy mini Kits (Qiagen, Germany) and miRNeasy serum/plasma (Qiagen, Germany) were used to extract miRs from serum samples. In addition, miRNeasy procedures reduce the chances of contamination with salt or phenols, that interfere with further processing. According to manufacturer’s protocol, 200 µl should be taken for RNA extraction.
Mirneasy serum plasma
Manufactured by Qiagen
Sourced in Germany
The MiRNeasy Serum/Plasma kit is a product designed for the isolation of total RNA, including small RNAs such as microRNAs, from serum or plasma samples. The kit provides a fast and efficient method for the extraction of high-quality RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Exoquick, by System Biosciences (2 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
Mirvana paris, by Thermo Fisher Scientific (1 mentions)
Qiacube, by Qiagen (1 mentions)
Trizol, by Qiagen (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Qiazol, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Monarch total rna miniprep kit, by New England Biolabs (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy serum plasma advanced, by Qiagen (1 mentions)
Mirnaeasy mini, by Qiagen (1 mentions)
9 protocols using mirneasy serum plasma
1
Serum microRNA Profiling for Tumors
2
Comparative Evaluation of miRNA Extraction Kits
3
Quantification of lncRNA POU5F1B Expression
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lncRNA was extracted from plasma using miRNeasy Serum Plasma (Qiagen), then transcription was reversed to cDNA (Takara) fluorescence using SYBR green (Takara, R0036A). PCR analysis of POU5F1B was calculated by 2–ΔΔ method, with GAPDH as control. Total RNA in cells was extracted using Trizol (Qiagen). The genetic sequences were: forward: 5′-GCCATACGGTCACAGAGCTT-3′, reverse: 5′-CCCCCACATAACTCATACGG-3′; GAPDH forward: 5′-GGA GTCCACTGGCGTCTT-3′, reverse: 5′-GAGTCCTTCCACGATACCAA-3′. Condition of POU5F1B RT-PCR: 94°C for 30 s; 57.5°C for 30 s for annealing; 72°C for 75 s for extension; and finally 72°C for 7 min for elongation, for 32 cycles.
4
Transcriptome Analysis of Valvular Heart Disease
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To describe the transcriptome profile of patients with VHD, microRNAs will be extracted from platelet-free plasma and frozen valve fragments [59 (link), 60 (link)]. The miRNeasy Serum/Plasma and miRNeasy Tissue/Cells kits will be used for miRNA extraction according to the manufacturers’ protocols (Qiagen, Hilden, Germany). Targeted and untargeted transcriptome analysis will be used to quantify dysregulated miRNAs [59 (link)–62 (link)]. High-throughput techniques will be used to sequence the extracted miRNAs. Integrative bioinformatics will be used to determine the mRNAs targeted by the altered miRNAs in RHD and AS and correlate them to proteomics and metabolomics biomarkers [59 (link)–62 (link)]. Transcriptomic biomarkers with a significant q value (q <0.05 will be considered important.
5
Plasma RNA Extraction from Murine Samples
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Blood was collected from P2-P6 or P60 mice in EDTA-coated tubes and cell-free plasma fractions (which may include clotting factors) separated by centrifugation (0.6 x g) at 4°C. Samples were processed immediately and evaluated for hemolysis by an individual blind to the sample identity using visual inspection against a white background or by comparing A414 nm of sample to buffer (NanoDropt™); samples with significant hemolysis were not further processed. Plasma fractions were lysed (QIAzol, Qiagen) in the presence of spike-in control RNA (cel-miR-39-3p, or ath-miR159a; Qiagen, 1.6 x 108 copies/ul), and RNA isolated (miRNeasy serum/plasma, Qiagen)
6
miRNA Extraction from Plasma
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miRNA extraction from 200 μL dissolved plasma was performed in accordance with the QIAGEN® miRNeasy Serum/Plasma kit procedure.
7
Optimized RNA Extraction from Serum
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Two hundred microliters of frozen-stored serum was thawed at 4°C, mixed with 1000 μl of QIAzol Lysis Reagent (Qiagen), and vortex-mixed at room temperature. After incubation for 5 minutes at room temperature, 3.5 μl of synthetic Caenorhabditis elegans miRNA (cel-miR-39, 1.6 × 10 8 copies/μl) (Qiagen) was added and mixed with the sample. Extraction then proceeded per the miRNeasy serum/plasma manufacturer's protocol (Qiagen). All samples were eluted in 14 μl of RNase-free water. The concentrations of the final RNA yield were measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific).
8
Comparative Assessment of RNA Extraction Kits
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RNA was extracted from either fresh or frozen plasma according to the manufacturer’s instructions using 6 commercially available kits; Kit A: miRNeasy Serum/Plasma (Qiagen, Germany), Kit B: miRNeasy Serum/Plasma Advanced (Qiagen, Germany), Kit C: Quick-cfRNA Serum & Plasma (Zymo Research, USA), Kit D: Isolate II miRNA (Bioline, Australia), Kit E: PureLink RNA Mini kit (Invitrogen, USA), Kit F: Monarch Total RNA Miniprep kit (New England BioLabs, USA). All extractions were performed in duplicate for each animal, except for the PureLink and Monarch kits due to sample and reagent availability. Following extraction and elution of RNA, samples were immediately frozen at −80 °C.
9
Exosomal miRNA Extraction from Serum
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From each participant, whole blood was taken and processed using centrifugation at 1900 × g for 10 min centrifuge. To remove cell debris, the supernatant was centrifuged at 4 °C and 16,000 × g for 10 min and then stored at -80 °C. Total miRNA was extracted from serum samples using a kit (miRNeasy Serum/Plasma; Qiagen Inc.) according to the manufacturer's instructions. Exosomes was extracted from serum samples by using the polymer precipitation method (ExoQuick; System Biosciences, Palo Alto). Then, exosomal RNA was extracted using a kit (miRNAeasy Mini: Qiagen Inc.).
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