Total RNA was isolated from whole blood collected in PAXgene Blood RNA Tubes using PAXgene Blood RNA Kit (PreAnalytiX) per manufacturer's instructions, and PBMCs using TRIzol (Thermo Fisher Scientific). Total RNA with high quality (RIN > 8) was used for cDNA library preparation using the TruSeq Stranded mRNA Library Preparation kit for NeoPrep (Illumina). Sequencing was performed on an Illumina HiSeq 3000 System in a 1 × 50 bp single read mode. Sequenced reads were mapped against the human reference genome (GRCh37) using TopHat v2.1.1 (7 (link)). Reads mapped to hemoglobin genes were removed from further analysis. Mapped reads were quantified using HTSeq (8 (link)). All the count data were normalized using TCC (9 (link)) and differentially expressed genes were detected using edgeR (10 (link)). Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) software (Qiagen), respectively. The original RNAseq data is uploaded and available online (Gene Expression Omnibus: GSE118901 ).
Paxgene blood rna kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PAXgene Blood RNA Kit is a laboratory equipment product designed for the stabilization, purification, and isolation of RNA from whole blood samples. It provides a standardized and reproducible method for the collection, storage, and processing of blood samples to obtain high-quality RNA for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hiseq 3000 system, by Illumina (1 mentions)
Truseq stranded mrna library preparation kit for neoprep, by Illumina (1 mentions)
Paxgene blood rna tubes, by Thermo Fisher Scientific (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Globinclear kit, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna library preparation kit, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Amplitaq gold polymerase, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Illustra gfx pcr dna and gel band purification kit, by GE Healthcare (1 mentions)
Paxgene blood rna tube, by BD (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Hiseq x ten, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ribo zero gold rrna removal kit, by Illumina (1 mentions)
Universal human reference rna, by Agilent Technologies (1 mentions)
Paxgene blood rna tube, by Qiagen (1 mentions)
Nanodrop nd 2000 uv spectrophotometer, by Thermo Fisher Scientific (1 mentions)
5 protocols using paxgene blood rna kit
1
Whole Blood and PBMC RNA-seq Profiling
2
Whole Blood RNA-Seq Protocol
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Total RNA was extracted from whole blood using PAXgene Blood RNA Kit (PreAnalytiX), followed by treatment with GLOBINclear™ kit (ThermoFisher Scientific) to remove unwanted globin mRNA. The remaining RNA product was used to prepare cDNA library using Illumina TruSeq Stranded mRNA library preparation kit (Illumina). RNAseq was performed by Beijing Genomics Institute using the Illumina Hiseq2000 with 75 base-pair (bp) paired-end reads to a depth of at least 30 million reads per sample.
RNAseq output was processed as previously described [76 (link)]. Read pairs were pre-processed to adjust base calls with phred <5 to ‘N’ and to remove read pairs where either end had fewer than 30 unambiguous base calls. This method indirectly removes read pairs containing mostly adaptor sequences. Read pairs were aligned to the human genome (hg19) using STAR (v2.3.1d) [77 (link)]. Gene counts were tabulated using htseq (v0.6.0) with the intersection-strict setting turned on and Ensembl gene annotations (GRCh37.74) used to map genomic locations to gene identifiers [78 (link)]. The edgeR (v3.36.0) package function, cpm, was used to calculate TMM-normalized counts-per-million (CPM) expression matrices [79 (link)].
RNAseq output was processed as previously described [76 (link)]. Read pairs were pre-processed to adjust base calls with phred <5 to ‘N’ and to remove read pairs where either end had fewer than 30 unambiguous base calls. This method indirectly removes read pairs containing mostly adaptor sequences. Read pairs were aligned to the human genome (hg19) using STAR (v2.3.1d) [77 (link)]. Gene counts were tabulated using htseq (v0.6.0) with the intersection-strict setting turned on and Ensembl gene annotations (GRCh37.74) used to map genomic locations to gene identifiers [78 (link)]. The edgeR (v3.36.0) package function, cpm, was used to calculate TMM-normalized counts-per-million (CPM) expression matrices [79 (link)].
3
Blood RNA Extraction and Sequencing Protocol
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Blood was collected in a PAXgene® Blood RNA tube (BD Biosciences, Franklin Lakes, NJ, USA), RNA was extracted using a PAXgene® Blood RNA kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reverse transcribed using a Superscript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, Inc.). Corresponding complementary DNA (cDNA) was amplified in a 50 µl reaction containing: 5 µl 10X AmpliTaq Gold Polymerase buffer, 5 µl dNTP (25 mM), 20 pmol (final concentration) specific exon forward/reverse primers (Table II ) and 1 µl AmpliTaq Gold Polymerase (all Thermo Fisher Scientific, Inc.). PCR cycling conditions were as follows: Initial denaturation at 95°C for 10 min; 25 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec; final extension of 72°C for 7 min. Amplicons were purified from the gel using an Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences, Chalfont, UK) and directly sequenced using a Big Dye Terminator v1.1 Cycle Sequencing kit (Thermo Fisher Scientific, Inc.).
4
Comprehensive Transcriptome Profiling of lncRNAs from Blood Samples
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Total RNA was extracted from peripheral blood using a PAXgene blood RNA kit (Thermo Scientific, USA) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and the RNA integrity number (RIN) was estimated using an Agilent 2100 bioanalyzer (Agilent Technologies, USA). Samples with RIN ≥ 7 were considered adequate for subsequent analysis. Two micrograms of RNA per sample was used for lncRNA library construction by a TruSeq Stranded Total RNA kit with Ribo-Zero Gold (cat. no. 15021048, Illumina, USA). Briefly, ribosomal RNA was removed using a Ribo-Zero Gold rRNA removal kit (Illumina), and rRNA-free RNA was reverse transcribed into complementary DNA (cDNA). After ligation of the terminal adapters, PCR amplification, and purification, the quality of the library was assessed using an Agilent bioanalyzer 2100 system. Then, the libraries were sequenced on the Illumina sequencing platform (HiSeq X Ten) with PE150 according to the manufacturer’s protocol. All data analysis was performed by OE Biotech Co. Ltd. (Shanghai, China).
5
Quantifying Human mRNA Expression
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Peripheral blood was collected in 2.5 ml PAXgene Blood RNA Tubes (PreAnalytiX; Qiagen, Hilden, Germany) for the mRNA analysis. Total RNA was extracted using a PAXgene Blood RNA Kit (PreAnalytiX) according to the manufacturer's instructions and was measured for quantity and purity using a NanoDrop® ND-2000 UV spectrophotometer (Thermo Scientific, Waltham, MA, USA). cDNA synthesis was performed with 500 ng of total RNA using an M-MLV Reverse Transcriptase system (200 U/µl; Mbiotech, Inc., Seoul, Korea), and cDNA was stored at −80°C until use. TaqMan gene expression assays (StepOnePlus™; Applied Biosystems, Foster City, CA, USA) were performed under the standard TaqMan protocol (10 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 60°C) in a 96-well microplate. We used primers and probes purchased commercially from Integrated DNA Technologies (Coralville, IA, USA) for PCR assays. As an endogenous control, 18S ribosomal RNA and the commercially available Universal Human Reference RNA (Agilent Technologies, Santa Clara, CA, USA), to reduce the batch effect, were used to calculate gene expression levels using the comparative CT method. The results were then log10-transformed to reduce the deviation from the normalized data in the qRT-PCR system prior to statistical analysis.
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