DNA and total RNA were extracted from fresh frozen non-neoplastic breast tissue samples from cohort-2 using the Allprep kit DNA/RNA/miRNA (Qiagen no. 80224). Nucleic acids were not isolated from normal tissue samples from cohort-1 as only a handful of samples exist. Since normal breast tissue is enriched in fatty tissue and stromal cells, as compared to the tumor, a section of normal breast tissue was stained with eosin and hematoxylin, before extraction, to ensure the presence of normal epithelial breast cells in the specimen. Thirty-five samples were chosen for continuation based on the presence of normal breast tissue, RNA quantity and RIN (RNA integrity number) value. DNA and total RNA was extracted from cohort-2 by the same method used for normal breast tissue [18 (link)], but total RNA from cohort-1 was extracted with Trizol, as described [19 (link)].
Allprep dna rna mirna kit
Manufactured by Qiagen
Sourced in Germany
The AllPrep DNA/RNA/miRNA kit is a laboratory product that allows for the simultaneous isolation of DNA, RNA, and microRNA from a single sample. It provides a comprehensive solution for extracting and purifying these three biomolecules from a variety of sample types.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (7 mentions)
Dneasy blood tissue kit, by Qiagen (6 mentions)
Hiseq 2500, by Illumina (6 mentions)
Qiashredder, by Qiagen (5 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Tissuelyser 2, by Qiagen (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Novaseq 6000, by Illumina (2 mentions)
2200 tapestation automated electrophoresis system, by Agilent Technologies (2 mentions)
Hiseq 2500 ultra high throughput sequencing system, by Illumina (2 mentions)
Purezol rna isolation reagent, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rna 6000 nano chip, by Agilent Technologies (2 mentions)
Ultra low attachment t25 flasks, by Corning (2 mentions)
Cell recovery solution, by Corning (2 mentions)
Sureselect human all exon v4 platform, by Agilent Technologies (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Prepit l2p, by DNA Genotek (2 mentions)
50 protocols using allprep dna rna mirna kit
1
Extraction of DNA and RNA from Breast Tissue
2
Extraction and Characterization of Breast Tumor RNA/DNA
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DNA and total RNA were extracted from fresh frozen breast tumors from patients in cohort 2 (n = 291) and from 6 normal breast tissue samples as well as 1x106 MCF-7 cells using Allprep kit DNA/RNA/miRNA (Qiagen no. 80224) according to protocol. The extraction from cohort 1 has been described [22 (link)] but in short, total RNA was extracted with Trizol and purified on an RNeasy column according to protocol. The quantity of DNA was measured by Nanodrop 1000 and the RNA quality was measured with Bioanalyzer 2100 RNA 6000 Nano kit (Agilent Technologies, cat. no. 5067–1511) according to protocol. The majority of tumors had RIN ≥ 8.
3
Transcriptome Analysis of Neonatal ECFCs
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Total RNA was extracted from neonatal ECFCs using the AllPrep DNA/RNA/miRNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. After determination of suitable RNA quality (RNA integrity number, RIN) scores via the RNA TapeStation system (Agilent, Santa Clara, CA, USA), libraries were prepared with the TruSeq stranded mRNA kit (Illumina, San Diego, CA, USA) by the Victorian Clinical Genetic Services (VCGS) Sequencing Service (Melbourne, Australia). Subsequently, sequencing was performed on the NovaSeq 6000 (Illumina) with 150 bp paired ends. Sequencing reads were aligned to the GRCh37 (hg19) human reference transcriptome using Bowtie (Langmead, 2010 ). Quantification of gene expression levels as reads per kilobase per million (RPKM) and counts was performed using MMSEQ (Turro et al., 2011 (link)). Reads/transcripts were normalized using DESeq2 (Love et al., 2014 (link)) and transcripts were annotated as protein coding or lincRNA using Biomart (Smedley et al., 2009 (link)). Only the subset of the gene biotype ‘lincRNA’ was considered for downstream analysis. Differentially expressed transcripts were identified by linear regression using DESeq2 with an unadjusted P‐value cut‐off of <0.05 considered significant.
4
RNA Extraction from Post-Selection Cells
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Post-selection cells were spun down and re-suspended in 350μl of RLTplus buffer with 1% beta-mercaptoethanl and transferred to 2ml tubes. Samples were stored at -80°C for batched RNA extraction. Homogenization of the sample was carried out using the QIAshredder (Qiagen). The AllPrep DNA/RNA/miRNA kit (Qiagen) was used for RNA extraction. DNase I was used during the extraction protocol to minimise DNA contamination. RNA was eluted into 35μl of RNase- free water. The RNA amount was quantified by Qubit analysis and the RNA samples stored at - 80°C for storage until ready for sequencing.
5
Quantification of Proviral DNA Load
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DNA was isolated using the Qiagen AllPrep DNA/RNA/miRNA kit and proviral DNA was quantified by real-time PCR using primers targeting either the BLV or HTLV-1 3′ region and RPS9 or Actin, respectively, for normalization (primers from Integrated DNA Technologies; Supplementary Data 5 ). Runs were performed in a 50 μl volume containing 1 μg of total DNA, primers and probe (200 nM concentration of each) in 1 × PCR buffer (Platinum Quantitative PCR SuperMix-UDG) (HTLV-1) or 10 μl containing 50 ng DNA and 1 × Universal PCR Master Mix, No AmpErase UNGa (ThermoScientific) (BLV). Thermocycling conditions were 10 min at 95 °C, followed by 50 cycles at 95 °C for 15 s and 60 °C for 1 min. Standard curves were generated using serial dilutions of DNA from the YR2 cell line (BLV, two proviral copies) or the Tarl2 cell line (HTLV-1, single proviral copy). Proviral load in % PBMCs=(Sample Average Quantity) × 2/(Sample RPS9 or Actin quantity) × 100. The YR2 chromosome that carries the BLV provirus integration appears to be duplicated.
6
Simultaneous DNA and RNA Extraction
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Extraction of total nucleic acids from 106 infected cells was performed with an AllPrep DNA/RNA/miRNA kit (Qiagen, Hilden, Germany), allowing us to simultaneously obtain the total DNA and RNA from any individual sample.
7
Transcriptome Profiling of Lung and Heart Tissues
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Lung and heart tissues were processed by slicing approximately 30 mg of tissue from each sample and homogenizing in Buffer RLT Plus for 4 min at 20 Hz using the TissueLyser II (Qiagen). Total RNA was extracted using the AllPrep DNA/RNA/miRNA kit (Qiagen). RNA was quantified with a Nanodrop 1000 spectrophotometer (Thermo Scientific) and quality-verified on a representative subset of 30 samples using the 2200 TapeStation Automated Electrophoresis System (Agilent Technologies). Samples showed RNA integrity numbers (RINs) that greatly exceeded requirements for the employed sequencing technologies, with RINs ≥ 8.8. RNA samples were then analyzed using the TempO-Seq Mouse Whole Transcriptome assay (BioSpyder) with resulting libraries sequenced using the HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina). Sequencing data were processed using the Tempo-SeqR pipeline (BioSpyder 2021 ), with QA/QC metrics including the average number of mapped reads in positive controls (i.e., requiring > 6×106 mapped reads), low signal-to-noise ratios (i.e., requiring total number of mapped reads in the positive control divided by the total number of mapped reads in negative controls > 20:1), and the percentage of mapped reads in positive control (i. e., requiring > 80%). Count data were aligned to the Ensemble database v98 and summarized as number of counts per gene.
8
RNA Isolation from Cell Lines and Tissues
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Total RNA, including miRNAs, was isolated from HPC0A07/03C cells using the AllPrep DNA/RNA/miRNA kit (Qiagen, Hilden, Germany) and from rats’ brains using PureZol RNA isolation reagents (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s protocols. RNA quantity and quality were assessed by evaluation of the A260/280 and A260/230 ratios using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
9
Analyzing Cerebral Gene Expression
10
Newborn Mouse Heart RNA Extraction
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RNA was extracted from 24 newborn B × C female whole hearts using the AllPrep DNA/RNA/miRNA kit (Qiagen, 80204). Eight 0 ppm females representing 6 litters, eight 1 ppm females representing 4 litters, and eight 50 ppm females representing 5 litters were used. Hearts stored at −80 °C were homogenized in the kit’s lysis buffer using a microtube homogenizer (Biospec 3110Bx Cell Disrupter 4800, BZ10124883). RNA was extracted from the hearts following the manufacturer’s protocol for 10–30 mg of starting material. RNA was suspended in nuclease-free water and quantified using a Nanodrop 2000. RNA purity and size integrity were determined at the NCSU Genomic Sciences Laboratory (GSL) using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies). All RNA samples had an RNA Integrity Number (RIN) ≥ 9.9.
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