Total RNAs were isolated from serum samples by use of Plasma/Serum RNA Purification Mini Kit (Norgen Biotek Corp., Thorold, Canada) according to the manufacturers’ protocols. Purified total RNAs were quantified using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Then, cDNAs were synthesized from 0.5 μg RNAs for further assay based on the instructions of PrimeScript™ RT reagent Kit (Takara, Tianjin, China). Real-time PCR was carried out in triplicate assay in accordance with the specifications of SYBR Green Mastermix kit (Takara, Tianjin, China). A total of 5 ng cDNA template was used for real-time PCR assay. Random primers were used in experiments. The expression of each lncRNA was represented as fold changes using the 2-ΔΔCTmethod and normalized to housekeeping gene GAPDH. Primer sequences used in validation of lncRNAs in this paper were listed in Table 4 .
Plasma serum rna purification mini kit
Manufactured by Norgen Biotek
Sourced in Canada
The Plasma/Serum RNA Purification Mini Kit is a laboratory equipment designed to extract and purify RNA from plasma and serum samples. It utilizes a silica-based membrane technology to capture and elute RNA, providing a reliable method for isolating high-quality RNA from these sample types.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Exoquick tc exosome precipitation solution, by System Biosciences (2 mentions)
Sybr green master mix kit, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Truseq small rna library preparation kit, by Illumina (1 mentions)
Nextseq, by Illumina (1 mentions)
Bluepippin dna size selection system, by Sage Science (1 mentions)
Taqman advanced mirna cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop lite, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Expression suite software v1, by Thermo Fisher Scientific (1 mentions)
Quantstudio software v1, by Thermo Fisher Scientific (1 mentions)
Agilent rna pico chip kit, by Agilent Technologies (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
Qev 70 columns, by Izon Science (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
12 protocols using plasma serum rna purification mini kit
1
Serum lncRNA Expression Profiling
2
Serum Small RNA Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from serum using the Norgen Biotek Corp. plasma/serum RNA purification mini kit. Sequencing libraries were prepared from RNA using the Illumina TruSeq small RNA library preparation kit according to manufacturer’s instructions with the following modifications. Fifteen cycles were used for the final library PCR amplification step. The sequencing libraries were purified with the BluePippin DNA size selection system by Sage Science using the 3% agarose gel cassettes, gating from 125–150 bp. Prior to sequencing, library purification was confirmed by running samples on an Agilent Bioanalyzer using high-sensitivity DNA chips. Purified libraries were sequenced in multiplex using either the Illumina MiSeq or NextSeq platforms. Samples were multiplexed such that approximately 5 million single-ended 80-nt reads were sequenced for each sample.
3
Extracellular Vesicle RNA Isolation and Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MiRNA isolation and purification of cell-derived EVs was done using the Plasma/Serum RNA Purification Mini Kit from NORGEN (Norgen Biotek Corporation, Therold, ON, Canada) according to the manufacturer supplementary protocol for EV RNA purification from EVs already isolated from precipitation methods. RNA concentration and purity were measured using the NanoDrop Lite spectrophotometer (Thermo Scientific®, Waltham, MA, USA) and served as template for cDNA synthesis using a TaqManTM Advanced miRNA cDNA Synthesis Kit (Applied Biosystems®, Foster City, CA, USA) according to the manufacturer protocol.
4
Exosome Isolation and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
S2‐013 and HPNE cells were lysed in lysis buffer [50 mm Tris (pH 7.4), 150 mm NaCl, 1 mm MgCl2, 0.5% NP‐40, a protease inhibitor cocktail tablet (Roche Applied Science, Penzberg, Germany), and phosphatase inhibitor cocktail (Nacalai, Kyoto, Japan)]. The exosomes in the cell lysates were precipitated using an ExoCap Exosome Composite kit (JSR Life Sciences, Tokyo, Japan) according to the manufacturer's recommendations. Exosomes in conditioned medium harvested from S2‐013 and HPNE cells were precipitated using ExoQuick‐TC exosome precipitation solution (System Biosciences) according to the manufacturer's recommendations. RNA was extracted from exosomes isolated from either cell lysates or cell culture medium using a Plasma/Serum RNA Purification Mini kit (Norgen BIOTEK, Thorold, ON, Canada) according to the manufacturer's instructions. The exosomal RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific, Fremont, CA, USA).
5
Exosomal RNA Extraction from Serum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exosomal RNA was extracted from all serum samples using a Plasma/Serum RNA Purification Mini kit (Norgen BIOTEK) according to the manufacturer's recommendations. The RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific).
6
Exosomal miRNAs Profiling by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from CM exosomes, obtained as above described, and exosome‐free control medium using the Plasma/Serum RNA Purification Mini Kit (Norgen Biotek, Canada) according to the manufacturer's instructions.
MiRNAs analysis in exosomal component was investigated by quantitative Real‐Time PCR (qRT‐PCR) using TaqMan Advanced miRNA Human Serum/Plasma Card (Applied Biosystem). This method allows evaluating the expression of 188 miRNAs, each of them in duplicate, with high sensitivity and specificity starting from a very low amount of total RNA input.
The analysis was performed by examining 2 replicates, by using RNAs extracted by two distinct CM‐exosome preparations compared with control medium. Comparative expression analysis was performed by QuantStudio Software v 1.3 and Expression Suite software v 1.3 (Applied Biosystems). Ath‐miR159a was used as exogenous control for normalization of data, and global normalization analysis was conducted as well. ΔΔCt method was applied to determine the relative miRNAs expression levels. Furthermore, manual analysis focused on PCR amplification plots profiles was performed, and miRNAs showing mean Ct less than 30 and relative quantification (RQ) values more than twofold (linear scale) were considered.
MiRNAs analysis in exosomal component was investigated by quantitative Real‐Time PCR (qRT‐PCR) using TaqMan Advanced miRNA Human Serum/Plasma Card (Applied Biosystem). This method allows evaluating the expression of 188 miRNAs, each of them in duplicate, with high sensitivity and specificity starting from a very low amount of total RNA input.
The analysis was performed by examining 2 replicates, by using RNAs extracted by two distinct CM‐exosome preparations compared with control medium. Comparative expression analysis was performed by QuantStudio Software v 1.3 and Expression Suite software v 1.3 (Applied Biosystems). Ath‐miR159a was used as exogenous control for normalization of data, and global normalization analysis was conducted as well. ΔΔCt method was applied to determine the relative miRNAs expression levels. Furthermore, manual analysis focused on PCR amplification plots profiles was performed, and miRNAs showing mean Ct less than 30 and relative quantification (RQ) values more than twofold (linear scale) were considered.
7
RNA Isolation and Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the samples were submitted to Norgen facilities for Next-Generation Sequencing (NGS). The whole RNA was isolated by using Plasma/Serum RNA Purification Mini Kit (Norgen Biotek, Thorold, ON, Canada). Ribogreen kit assay (Thermofisher, Waltham, MA, USA) was performed for the quantification in a microplate reader, at 260 nm, using 1 μL of RNA, and the quality was measured with the Agilent RNA Pico chip kit (Agilent Technologies, Santa Clara, CA, USA).
8
Efficient Plasma/Serum cfRNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell-free RNA was isolated using the Plasma/Serum RNA Purification Mini Kit (Cat. 55,000, Norgen Biotek, Canada), which isolates both cfRNA and vesicle-encapsulated RNA, following the manufacturer’s instruction with slight modifications. Briefly, plasma/serum samples were allowed to thaw on ice and then 200 µL of plasma/serum cleared at 16,000 × g for 2 min at 4 °C and the supernatant transferred into a new tube. The precleared plasma/serum was then combined with 3 volumes (600 µL) of Lysis buffer A, containing 0.01% β-mercaptoethanol and mixed by vortexing. To one volume of lysate (800 µL), an equal amount of absolute ethanol was added and mixed by vortexing for 5 s. The lysate was then loaded onto a mini column and allowed to stand at room temperature for 10 min for RNA binding. The columns were then centrifuged at 6000 rpm for 3 min. The columns were then washed 3× with 400 µL of wash buffer A and dried by centrifugation at 13,000 rpm for 2 min. RNA was eluted in 25 µL of elution buffer A after incubation on the column for 15 min. The RNA concentration was measured using a Quantus fluorometer (Promega, Madison, Fitchburg WI, USA), and samples were stored at −80 °C until analyzed.
9
Extracellular Vesicle RNA Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EVs were isolated from 1 mL of plasma by SEC columns of polysaccharide resin (qEV 70 columns; IZON, Christchurch, New Zealand) following the company’s protocol. The EV-enriched fractions were collected. Subsequently, RNA was extracted from EVs by using the Plasma/Serum RNA Purification Mini Kit (Cat. 56100, Norgen Biotek, ON, Canada) as indicated in the manufacturer’s protocol. The extracted RNAs were qualitatively evaluated with the Bioanalyzer 2100 instrument (Agilent Technologies, Milan, Italy) using RNA6000 pico chips.
10
Quantifying GPC1 mRNA in PANC-1 EV Serum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GPC1 mRNA levels in PANC‐1 EVs spiked healthy donor serum were quantified using qRT‐PCR. Total RNA from 150 µL of calibration samples made above was isolated using a plasma/serum RNA purification mini kit (#55000, Norgen Biotek Corp.) according to the manufacturer's instructions. cDNA was then synthesized from total RNA using a High‐Capacity cDNA reverse transcription kit (#4368814, Applied Biosystems). Subsequently, the target mRNA expression was quantified using a TaqMan Gene Expression assay (Hs00892476_m1, ThermoFisher Scientific) on a real‐time PCR instrument (Applied Biosystems).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!