RNeasy Serum/Plasma Kit (217184, Qiagen) was used to isolate the RNA from the EVs and HDL particles according to the manufacturer's instructions. Before isolation, 140 μL of filtered (0.2 μm) PBS (pH 7.4) was used to obtain the recommended starting volume of 200 μL. Spike‐in cel‐miR‐39‐3p (MS00019789, Qiagen) was used as an internal control (housekeeping miR).
Rneasy serum plasma kit
Manufactured by Qiagen
Sourced in United States
The RNeasy Serum/Plasma Kit is a laboratory equipment product designed for the purification of total RNA from serum or plasma samples. It provides a reliable and efficient method for extracting high-quality RNA from these types of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Spike in cel mir 39 3p, by Qiagen (2 mentions)
Rneasy ffpe kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Sybr qpcr mix, by Takara Bio (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Improm 2 reverse transcriptase, by Promega (1 mentions)
Mx3000p instrument, by Agilent Technologies (1 mentions)
Eastep total rna extraction kit, by Promega (1 mentions)
Bulge loop mirnaqrt pcr primer kit, by RiboBio (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Reverse transcriptase, by Promega (1 mentions)
Sybr green qpcr supermix udg, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rnalater ice solution, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
8 protocols using rneasy serum plasma kit
1
RNA Isolation from Extracellular Vesicles
2
Exosomal RNA Isolation and Analysis
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Total RNA derived from CSF, serum and medium were isolated from the prepared exosomes using an RNeasy serum/plasma Kit (Qiagen, USA), as recommended by the manufacture’s protocol. The quality and yield of each RNA sample was measured using a 2100 Bioanalyzer (Agilent Technologies, Sata Clara, CA) and NanoDrop (NanoDrop Technologies, Houston, TX). RT-qPCR was used to detect both cellular and exosomal mRNAs/miRNA. The primers for GAPDH, miR-21, PTEN, RECK and PDCD4 were purchased from Ambion. RT-PCR parameters were set following the manufacturers’ instructions. The exosomal miR-21 data were normalized to GAPDH and referred as the ratios to the scramble control when being used.
3
Quantitative Analysis of Circulating RNA
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Total RNA was extracted from tissue samples using TRIzol reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), according to the manufacturer's protocol. Plasma cfRNA was extracted from 200 µl plasma using the RNeasy Serum/Plasma kit (Qiagen GmbH), according to the manufacturer's protocols. cDNA was synthesized and amplified using ImProm-II™ reverse transcriptase (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. The primer sequences are indicated in Table IV . qPCR was subsequently performed using the SYBR qPCR mix (Takara Biotechnology Co., Ltd., Tokyo, Japan) with an Mx 3000p instrument (Agilent Technologies Inc., Santa Clara, CA, USA), according to the manufacturer's protocols. Amplification of the cDNA was confirmed using melting curve analysis. cDNA was reverse transcribed from 20 µl plasma cfRNA and the following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 5 min; 45 cycles of denaturation at 95°C for 10 sec and annealing and extending at 60°C for 20 sec. The relative expression level of each RNA was quantified using the 2−∆∆Cq method (19 (link)) with the 18S rRNA gene as the endogenous control for data normalization, since its expression level does not significantly differ in the plasma samples of patients with GC and healthy participants (20 (link),21 (link)).
4
lncRNA and miRNA Expression Profiling
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Total RNAs of rabbit and human VSMCs were isolated using Eastep™ Total RNA Extraction Kit (Promega, Beijing, China). Blood samples were extracted by RNeasySerum/Plasma Kit (Qiagen, Hilden, Germany), and RNeasy FFPE Kit (Qiagen, Hilden, Germany) for FFPE total RNA. Amplification of lncRNA and miRNAs was performed by GoScript™ Reverse Transcription System (Promega, Madison, WI, USA) and Bulge-LoopTM miRNA qRT-PCR Primer Kit (RiboBio, Guangzhou, China) respectively. The qPCR was performed on Applied Biosystems® 7500 Real-Time PCR System with SYBR green method (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as endogenous reference for lncRNA expression while U6 for miRNAs expression.
5
Quantitative RNA Isolation and Analysis
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TRIzol reagent (Invitrogen) was utilized to isolate total RNA from NPC tissues and cells. The RNeasy serum/plasma kit (QIAGEN GmbH) was applied to extract total RNA from serum samples. The RNA quality and amount were evaluated by a NanoDrop 3300 spectrophotometer (Thermo Scientific). Reverse transcriptase (Promega) was used to perform reverse transcription. SYBR Green qPCR Super Mix-UDG (Thermo Fisher) was employed to conduct Quantitative real-time PCR (qRT-PCR). β-tubulin was utilized as the normalization control. Specific primers are shown in Supplementary Table S4 .
6
Extraction of Total RNA from FFPE and Plasma Samples
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Formalin-fixed, paraffin-embedded tissue samples were cut into 10-µm-thick sections, and total RNA was extracted from tumor and normal gastric mucosa in each patient using RNeasy FFPE kit (Qiagen, Valencia, CA), according to the manufacturer’s protocol. In plasma samples, total RNA was extracted from 100 µL plasma using RNeasy Serum/Plasma kit (Qiagen), according to the manufacturer’s protocol.
7
RNA Extraction from HCC Cells and Tissues
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Total RNA was extracted from HCC cell lines HepG2 and Huh7 and immortalized normal liver PH5CH cells using Qiagen miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA was also extracted from two Grade-2 HCC tumor tissues (HCC103T, HCC105T, BioChemed) and a normal liver tissue using mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). All kits were used by following manufacturer guidelines. Tumor samples corresponding to two plasma samples (HCC103P, HCC105P, BioChemed) were investigated by RNAseq and one of them (HCC103T) was also studied by RT-qPCR. 100 mg tissue treated with RNAlater-ICE solution (Invitrogen, Carlsbad, CA, USA) was homogenized in a glass homogenizer, worked-up following kit protocol, and RNA eluted in 100 μl RNase-free water. Plasma (3 ml) from HCC101P (BioChemed) was spun at 2,000g for 15 minutes to remove cellular debris and supernatant was collected. Total RNA from extracellular vesicles (EVs) was extracted using the ExoMir Kit (BIOO Scientific Corp., Austin, TX, USA). RNA from the remaining EV-free flow-through plasma was extracted using the Qiagen RNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). RNA from plasma EVs, EV-free plasma, and liver tissues was sequenced at TJU as described above.
8
RNA Isolation from Extracellular Vesicles
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RNeasy Serum/Plasma Kit (217184, Qiagen) was used to isolate the RNA from the EVs and HDL particles according to the manufacturer's instructions. Before isolation, 140 µl of filtered (0.2 µm) PBS (pH 7.4) was used to obtain the recommended starting volume of 200µl. Spike-in cel-miR-39-3p (MS00019789, Qiagen) was used as an internal control.
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