We isolated total RNA of 461 subjects from serum samples using Qiagen miRNeasy Serum/Plasma extraction kit and QIAcube automation. All samples were quantified using the Nanodrop spectrophotometer prior to plating with the RNA concentration 30.97 ± 20.97 ng/μl (see Additional file 1 : Fig. S1). We built the small RNA-Seq libraries by Norgen Biotek Small RNA Library Prep Kit and sequenced on the Illumina NextSeq 500 platform at 51 bp single end reads. The sequencing data was deposited in the Gene Expression Omnibus with the accession number GSE134897 [13 (link)]. COMPSRA was employed to evaluate the read quality and trim adapters [22 (link)]. Sequencing reads with quality score lower than 20 were removed. The qualified reads were aligned to human genome hg38 by STAR (v2.7.10b) [23 (link)] and miRNAs were annotated by COMPSRA on the basis of miRbase [24 (link)].
Mirneasy serum plasma extraction kit
Manufactured by Qiagen
Sourced in Germany, United States
The MiRNeasy Serum/Plasma extraction kit is a product designed to isolate and purify total RNA, including microRNA (miRNA), from serum and plasma samples. The kit utilizes a phenol-based extraction method to capture the full complement of RNA species present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Miscript 2 rt kit, by Qiagen (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Small rna library prep kit, by Illumina (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Miscript syber green pcr kit, by Qiagen (1 mentions)
Rt2 sybr green pcr kit, by Qiagen (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Steponeplus machine, by Thermo Fisher Scientific (1 mentions)
Human serum plasma mirna pcr array, by Qiagen (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Mirneasy serum plasma spike in control, by Qiagen (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
7 protocols using mirneasy serum plasma extraction kit
1
Serum Small RNA Sequencing Protocol
2
Serum miRs Expression Profiling
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Blood samples were collected for the serum miRs assay just prior biopsy, and miR-200b, miR-21, and miR-29b were measured for all patients and control groups. Total RNA was extracted using a miRNeasy serum/plasma extraction kit (Qiagen, Valencia, CA, United States) using QIAzol lysis reagent according to the manufacturer’s instructions. RNA quality was determined using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, United States). Reverse transcription (RT) was carried out on 100 ng of total RNA in RT reactions in a final volume of 20 μL (incubated for 60 min at 37 °C and 5 min at 95 °C) using a miScript II RT Kit (Qiagen) according to the manufacturer’s instructions. Serum expression levels of mature miRNAs, miR-200b, miR-29b, and miR-21, were evaluated using miScript miRNA PCR primer assays and a miScript SYBER green PCR kit (Qiagen) according to the manufacturer’s protocol. The housekeeping miRNA SNORD68 was used as the internal control.
3
Serum lncRNA Quantification Protocol
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Materials used to execute this work include; one- MiRNeasy Serum/Plasma extraction kit (Qiagen, Valencia, CA, USA) was used for total RNA extraction from serum. Two- The RT2 First Strand Kit (Qiagen, Valencia, CA, USA) was used for reverse transcription to produce cDNA. Three- The RT2 SYBR Green PCR kit (Qiagen, Maryland, USA) was used to perform qRT-PCR along with specific primers supplied by Qiagen, (Valencia, CA, USA); for IFNG-AS1 (Catalog no; 330701LPH20079A, Accession no, ENST00000536914.0) and GAS5 (Catalog no; 330701LPH11340A, Accession no, NR_002578.2) and we used GAPDH primer (Catalog no: 330701 LPH31725A, Accession no: ENST00000496049.0) to standardize the expression pattern and quantify the target long non-coding RNAs.
4
Serum miRNA Purification Protocol
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According to the manufacturer's protocol; total miRNA purification from 200 μl of serum was performed with the miRNeasy‐serum/plasma extraction kit (Qiagen, Hilden, Germany). Samples were eluted with RNase‐free water for subsequent cDNA synthesis and stored at −80°C.
5
miRNA Profiling of Serum/Plasma Samples
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Total RNA was extracted with the miRNeasy Serum/Plasma extraction kit (Qiagen, Redwood City, CA) and purified using RNeasy MinElute Spin Columns. A total of 250 ng RNA was then reverse transcribed with a miScript II RT kit (Qiagen). miRNA expression profile was then measured using Qiagen’s Human Serum & Plasma miRNA PCR Array. Quantitative PCR was performed using a StepOnePlus machine (Applied Biosystems) per the manufacturer’s instructions. miRNA array data were analyzed using Ingenuity pathway analysis software (Qiagen) and DIANA-mirPath.
6
Profiling Circulating miRNA in Serum
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A small RNA fraction was extracted from 200µl of serum samples, using a miRNeasy Serum/Plasma extraction kit (QIAGEN, Hilden, Germany). C. elegans miR-39 was used as an RNA spike-in to normalize the gene expression analysis (miRNeasy Serum/Plasma Spike-in Control, QIAGEN, Hilden, Germany).
The identification and quantification of aberrantly expressed miRNAs, in the pool of samples form PR, NR and HR groups, was performed by using a large-scale quantification PCR-Array platform (miScript® QIAGEN, Helden, Germany) on a ViiA7 apparatus (Thermo-Fisher, Massachusetts, USA). We analyzed the data using a PCR Array Data Analysis Web Portal software (http://pcrdataanalysis.sabiosciences.com/mirna ).
The identification and quantification of aberrantly expressed miRNAs, in the pool of samples form PR, NR and HR groups, was performed by using a large-scale quantification PCR-Array platform (miScript® QIAGEN, Helden, Germany) on a ViiA7 apparatus (Thermo-Fisher, Massachusetts, USA). We analyzed the data using a PCR Array Data Analysis Web Portal software (
7
Quantitative Analysis of miRNA Biomarkers
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Total miRNA purification was performed with the miRNeasyserum/plasma extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Samples were eluted with RNasefree water for subsequent cDNA synthesis and stored at -80°C. The microRNA quantification was performed by qRT-PCR (Reverse Transcription coupled to Real-Time Polymerase Chain Reaction). Total RNA was reverse transcribed using a miScript II RT Kit (Qiagen, Hilden, Germany) in a StepOne Plus (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. Quantification of miR-155 (Cat. No. 00031486) and miR-483 (Cat. No. 00009758) mature miRNAs were determined using the miScript SYBR Green PCR kit (Qiagen, Hilden, Germany) and SNORD68 (Cat. No. 00033705) as normalizer. PCR reactions were each performed in a final volume of 15µl in the StepOne Plus thermocycler. The Melting Curve was performed to observe the specificity of reactions. Relative quantification was obtained using the Pfaffl method, which describes the use of reaction efficiencies obtained using LinReg PCR software. 12 Statistical tests were performed using GraphPad Prism. Data were examined for normality. Mann-Whitney U and Student's t-test were used to examine the statistical difference in between groups respect to miRNA expression. Statistical significance was set at p< 0.05.
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