A total of 0.5–1.5 mL of whole blood was used for white blood cells (WBC) RNA extraction. WBC were immediately separated by centrifugation using EL buffer (QIAamp RNA Blood Mini kit, Qiagen, Hilden, Germany). After cell separation, the WBC were collected, preserved in RNAlater (Qiagen) and stored at 10 °C until RNA extraction. WBC were lysed with RLT buffer, and the total RNA was extracted using QIAamp® RNA Blood Mini kit (Qiagen). RNA purity analysis and quantification were performed using the NanoVue spectrophotometer (GE Heathcare Life Sciences, Marlborough, MA). RNA quality was assessed on the Agilent BioAnalyzer 2100 (Agilent, Santa Clara, USA). All RNA samples were stored at − 80 °C until used in hybridization experiments.
Qiaamp rna blood mini kit
Manufactured by Qiagen
Sourced in Germany, United States, Japan, United Kingdom, Netherlands, China, Australia, France, Canada, Spain
The QIAamp RNA Blood Mini Kit is a laboratory equipment designed to efficiently extract and purify RNA from whole blood samples. It utilizes a silica-based membrane technology to capture and concentrate RNA, while effectively removing contaminants and inhibitors. The kit provides a reliable and consistent method for obtaining high-quality RNA suitable for downstream applications, such as qRT-PCR, Northern blotting, and microarray analysis.
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Lab products found in correlation
Quantitect reverse transcription kit, by Qiagen (14 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Primescript rt reagent kit, by Takara Bio (13 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (12 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (10 mentions)
Iscript cdna synthesis kit, by Bio-Rad (10 mentions)
Rnase free dnase set, by Qiagen (9 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (8 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (7 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Quantinova sybr green pcr kit, by Qiagen (7 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Qiaamp dna blood mini kit, by Qiagen (6 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (5 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (5 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (5 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (5 mentions)
374 protocols using qiaamp rna blood mini kit
1
RNA Extraction from White Blood Cells
2
RNA Extraction and cDNA Synthesis
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Ribonucleic acid (RNA) was extracted using QIAamp RNA blood mini kits (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Samples were extracted on the same day. The final RNA concentration was determined using a spectrophotometer (Nanodrop 2000, Therom Fisher, Walthm, USA) and RNA purity was verified by an average A260/ A280 ratio of 1.98 (range, 1.97–2.01). RNA was reverse transcribed to complementary deoxyribonucleic acid (cDNA) using a high-capacity cDNA reverse transcription kit (Applied Biosystems®, Branchburg, New Jersey, USA) in a final volume of 20 µl. Negative control samples were included in each set of reactions. Reactions were incubated at 25 °C for 10 min, followed by 37 °C for 120 min and final denaturation at 85 °C for 5 min. The reaction was carried out in the Bio-Rad thermal cycler (Life Science Research). cDNA was stored at − 20 °C.
3
Cytokine mRNA Expression Profiling
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To investigate the effect of encapsulated calcitriol on pro- and anti-inflammatory cytokine mRNA gene expression, RNA was extracted using QIAamp RNA Blood Mini kits (Qiagen NV) according to the manufacturer’s protocol. First, 100 ng of total RNA was transcribed into cDNA using the following mix: MgCl2 solution 25 mM, 10× PCR buffer, Oligo 50 μM, RNAse inhibitor, RT enzyme, and Ultrapure dNTP mix (Thermo Fisher Scientific). RT-qPCR reactions were run in duplicates containing SYBR Green I Master Mix (Hoffman-La Roche), ddH2O, and target forward/reverse primers, under the following conditions: preincubation at 95°C for 10 minutes followed by cycled amplification at 95°C for 10 seconds, with primer-dependent annealing temperature for 20 seconds, and 72°C for 5 seconds for 50 cycles. RT-qPCR was carried out using a LightCycler® 480 instrument. (Hoffman-La Roche). NormFinder® software was used to determine the stable housekeeping gene.40 (link) The relative mRNA concentration in each sample was calculated by using the second derivative max method. The target gene expression level of each sample was divided by the associated housekeeping gene GAPDH expression level. The mRNA ratios of the target gene/housekeeping gene were normalized to the untreated control. All primers and annealing temperatures are listed in Table 2 .
4
Quantification of IL1R2 mRNA Expression
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Venous blood samples were collected into BD PAXgene Blood RNA tube (Becton, Dickinson and Company, NJ, USA) from patients before premedication and after blood transfusion (within 24 h post transfusion or in the first 24 h of ATRs). Blood samples were stored at − 20 °C before RNA extraction. Total RNA was isolated using QIAamp RNA Blood Mini Kits (Qiagen, Nanjing, China) following the protocol from manufacturers. RNA samples were reversely transcribed into the first-strand cDNA and double-strand DNA. The amplification of IL1R2 mRNA was performed using an SYBR Green Master Mix kit (Vazyme, Nanjing, China) and the specific primers (IL1R2 forward: 5′-ACCGCTGTGTCCTGACATTT− 3′ and reverse: 5′-GGAAGAGCGAAACCCACAGA-3′). RT-PCR analysis was conducted according to the reaction conditions: 95 °C for 4 min; 40 cycles of 95 °C for 20 s, 58 °C for 30 s and 72 °C for 20 s; and 72 °C for 5 min. The relative expression level of IL1R2 mRNA was calculated using the 2−△△Ct method. GAPDH was used as the internal reference gene.
5
Transcriptional Profiling of Fracture-Induced Spinal Cord Injury
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Total RNA was isolated from was extracted from human whole blood of 16 patients with fractures complicated with SCI, 16 patients with fractures but no SCI and 8 normal adults, using QIAamp RNA Blood Mini Kits (Qiagen, catalog number 52304) according to the modified protocol of manufacturers. All cDNA generated from reverse transcription [PrimeScript RT Reagent Kit (Perfect Real Time) (Takara Bio)] was used for quantitative PCR analysis by ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, United States) and SYBR Premix Ex Taq (Tli RNaseH Plus) PCR Kit (Takara Bio). The relative expression levels of eight key genes (YY1, CEBPB, HAVCR2, LGALS9, MTOR, RPS6, RPS6KB1, RPS6KB2) and reference gene were determined by the 2–ΔΔCt method. Kruskal–Wallis test was used to identity the statistical difference of gene expression among groups.
Additionally, since the platform effect and batch effect could inevitably affect the results of the differential genes, two Affy Primeview dataset (GSE82152 and E-MTAB-5151) including normal peripheral blood samples were used as the control group for differential expression analysis. The limma package was also used to find differential expression genes (DEGs) after normalization between two batches of data (Ritchie et al., 2015 (link)). The standard of DEGs was an absolute log fold change greater than 1 and FDR P-value < 0.05.
Additionally, since the platform effect and batch effect could inevitably affect the results of the differential genes, two Affy Primeview dataset (GSE82152 and E-MTAB-5151) including normal peripheral blood samples were used as the control group for differential expression analysis. The limma package was also used to find differential expression genes (DEGs) after normalization between two batches of data (Ritchie et al., 2015 (link)). The standard of DEGs was an absolute log fold change greater than 1 and FDR P-value < 0.05.
6
Analyzing Activin A Expression by qRT-PCR
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To analyse activin A expression, mRNA levels of the β A subunit of activin A, INHBA, were determined using real‐time RT‐PCR. Total RNA was prepared from cultured cells using QIAamp RNA Blood Mini Kits (Qiagen). RNA aliquots (1 μg/20 μL) were reverse‐transcribed for 1 hour at 37°C using random hexamers (Promega, Mannheim, Germany) and High‐Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, California), in the presence of 1 u/μL of RNase inhibitor (Promega). cDNA quality was verified by amplifying the housekeeping gene, GAPDH. Gene expression (50 ng cDNA/20 μL) was quantified by real‐time PCR (Applied Biosystems 7500 Real‐Time PCR system).
7
Quantification of Vars2 Expression in Blood
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Whole RNA from peripheral blood samples was extracted with QIAamp RNA Blood Mini kits (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. RNA concentration and purity were measured with a spectrophotometer (NanoDrop, Thermo Fisher, Waltham, USA). Extracted RNA was reverse transcribed, and its concentration was measured. The relative expression of Vars2 with respect to GAPDH was calculated with the 2-ΔΔCt method and transformed with the log2FC formula. Primer design for Vars2 was performed with the NCBI Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast ).12
8
DNAH11 Transcript Variant Analysis
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Total RNA was extracted from 1–3 mL EDTA-anticoagulated whole blood using QIAGEN QIAamp RNA Blood Mini Kits according to manufacturer instructions. The extracted total RNA was reverse-transcribed to cDNA using Invitrogen Superscript Enzyme mix according manufacturer instructions. M13-tailed PCR primers flanking exon 65 (DNAH11-e64a-F1, 5′-TGTAAAACGACGGCCAGT AGAATGTCCACCGAAAATGC-3′; DNAH11-e64b-F2, 5′-TGTAAAACGACGGCCAGT TCCATTCCACTAACCGAAGG-3′; DNAH11-e66-R1/2, 5′-CAGGAAACAGCTATGACC TGCCAATTTTGTGTGAAGGA-3′) and primers flanking the termination codon (DNAH11-e82-F1, 5′-TGTAAAACGACGGCCAGTCCGTGGACAGACAAGAAACC-3′ and DNAH11-3′UTR-R1, 5′-CAGGAAACAGCTATGACCGACTTTCACTCTAAGGATAGCGTTG-3′) were used to PCR-amplify and Sanger-sequence the cDNA. cDNA product was visualized using the Agilent 4200 Tapestation system. Soft Genetics Mutation Surveyor (v5.1.2) was used to analyze Sanger sequence data, which was aligned against the DNAH11 NCBI Reference Sequence NM_001277115. Alamut Visual v2.11 was used to assess the transcript variants, including splicing effect prediction with SpliceSiteFinder-like, MaxEntScan, NNSPLICE, and GeneSplicer (Shapiro and Senapathy 1987 (link); Reese et al. 1997 (link); Pertea et al. 2001 (link); Yeo and Burge 2004 (link)).
9
Whole Blood RNA Isolation and Gene Expression Analysis
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Total RNA was isolated from cultured whole blood after erythrocyte lysis using QIAamp RNA Blood Mini kits (Qiagen, Valencia, CA). Genomic DNA was removed with RNase-free DNase (Qiagen). Reverse transcription employed High Capacity cDNA Reverse Transcription kits (Life Technologies, Foster City, CA). Real-time PCR was performed using TaqMan gene expression assays (Life Technologies). In order to minimize the possibility of genomic DNA expression, probes that span exon junctions were chosen whenever available from the manufacturer. No reverse transcription controls were used to confirm the absence of genomic DNA amplification for all gene expression assays. Beta actin served as normalization control and was multiplexed into all reaction wells. In order to assure noninterference of the housekeeping gene with the target genes, all gene expression assays were first pretested by comparing their amplification efficiencies as single-plex assay with their amplification efficiencies when multiplexed with the housekeeping gene. Reactions were run on a StepOne Plus Real-time PCR system (Life Technologies) using TaqMan Fast Advanced Master Mix (Life Technologies). Data were analyzed using the delta-delta threshold cycle (ΔΔC T ) method (23) .
10
RNA Extraction and qRT-PCR Protocol
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RNA extraction and quantitative reverse transcription PCR was carried out as detailed previously (Elkady et al., 2015) . Briefly, cells were plated and treated with indicated concentrations as aforementioned. 24-post treatment, both floating and adherent cells were collected, and pelleted by centrifugation at 700 g for 5 min. total ribonucleic acid (RNA) was extracted, reverse transcribed and amplified by polymerase chain reaction (PCR) using QIAamp® RNA Blood Mini Kits (QIAGEN) following manufacturer's instructions. Expression of tested genes was examined by PCR method using gene-specific primers.
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