C. japonicum was obtained from the National Institute of Biological Resources, Korea. The flower, leaf, and root tissues were immediately dissected and grinded in liquid nitrogen for the RNA extraction. The total RNA was extracted using Hybrid-R (Geneall, PN: 3033522) according to the manufacturer’s instructions. The total RNA was further treated with RNase-free DNase I (TaKaRa, Tokyo, Japan) and purified on an RNA-purification column (Qiagen, Valencia, CA, USA) to eliminate possible gnomic contamination. The RNA quality was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and RNA samples with RNA integrity number values above eight were used for the subsequent cDNA synthesis. The DNA was sheared with an average of 500 bp fragment sizes. The TruSeq Library Preparation Kit (Illumina Inc., San Diego, CA, USA) was used to construct the DNA library according to the manufacturer’s protocol. The DNA libraries were sequenced with 150-bp paired-end sequencing using an Illumina Hiseq2500. The quality of the constructed libraries was confirmed by a LabChip GX system (PerkinElmer, Waltham, MA, USA).
Rna purification column
Manufactured by Qiagen
Sourced in United States
The RNA-purification column is a laboratory equipment used for the isolation and purification of ribonucleic acid (RNA) from biological samples. The core function of this column is to efficiently capture and extract RNA molecules from complex mixtures, allowing for their subsequent analysis and downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Bead ruptor 12, by Omni International (2 mentions)
Cfx96 real time instrument, by Bio-Rad (2 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Hybrid r, by GeneAll (1 mentions)
Truseq library preparation kit, by Illumina (1 mentions)
Labchip gx system, by PerkinElmer (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Rnase free dnase, by Takara Bio (1 mentions)
Sureselect strand specific rna library kit, by Agilent Technologies (1 mentions)
Multi beads shocker, by Yasui Kikai (1 mentions)
Sodium cyanoborohydride, by Merck Group (1 mentions)
Lithium perchlorate, by Merck Group (1 mentions)
Rnase free dnase, by Thermo Fisher Scientific (1 mentions)
Hiseq 1500 machine, by Illumina (1 mentions)
12 protocols using rna purification column
1
RNA Extraction and Sequencing of Campanula japonicum
2
Quantification of SARS-CoV-2 RNA in Lung Tissue
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Viral RNA was isolated from lung tissue and subsequently amplified and quantified in a reverse transcription (RT)-qPCR reaction. Lung tissue was extracted at day 5 post infection and placed in 1 mL of trIzol reagent (Invitrogen). The samples were then homogenized using a Bead Ruptor 12 (Omni International). Tissue homogenates were then spun down and the supernatant was added to an RNA purification column (Qiagen). Purified RNA was eluted in 60 μL of DNase-, RNase-, endotoxin-free molecular biology grade water (Millipore). RNA was then subjected to reverse transcription and quantitative PCR using the CDC’s N1 (nucleocapsid) primer sets (Forward 5′-GAC CCC AAA ATC AGC GAA AT-3′; Reverse 5′-TCT GGT TAC TGC CAG TTG AATCTG-3′) and a fluorescently labeled (FAM) probe (5′-FAM-ACC CCG CAT TAC GTT TGGTGG ACC-BHQ1-3′) (Integrated DNATechnologies) on a BioRad CFX96 Real-Time instrument. For quantification, a standard curve was generated by diluting 2.5X106 PFU RNA equivalents of SARS-CoV-2. Every run utilized eleven 5-fold serial dilutions of the standard. SARS-CoV-2-negative mouse lung RNA and no templates were both included as negative controls for the extraction step as well as the qPCR reaction.
3
SARS-CoV-2 Viral Load Quantification
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Viral RNA was isolated from lung tissue and subsequently amplified and quantified in a reverse transcription (RT)-qPCR reaction. Lung tissue was extracted at day 5 post infection and placed in 1 mL of trIzol reagent (Invitrogen). The samples were then homogenized using a Bead Ruptor 12 (Omni International). Tissue homogenates were then spun down and the supernatant was added to an RNA purification column (Qiagen). Purified RNA was eluted in 60 μL of DNase-, RNase-, endotoxin-free molecular biology grade water (Millipore). RNA was then subjected to reverse transcription and quantitative PCR using the CDC’s N1 (nucleocapsid) primer sets (Forward 5’-GAC CCC AAA ATC AGC GAA AT-3’; Reverse 5’-TCT GGT TAC TGC CAG TTG AATCTG-3’) and a fluorescently labeled (FAM) probe (5’-FAM-ACC CCG CAT TAC GTT TGGTGG ACC-BHQ1–3’) (Integrated DNATechnologies) on a BioRad CFX96 Real-Time instrument. For quantification, a standard curve was generated by diluting 2.5X106 PFU RNA equivalents of SARS-CoV-2. Every run utilized eleven 5-fold serial dilutions of the standard. SARS-CoV-2-negative mouse lung RNA and no templates were both included as negative controls for the extraction step as well as the qPCR reaction.
4
RNA Extraction and Library Preparation for P. japonicus
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The frozen samples of P. japonicus were powdered using a Multi Beads Shocker (Yasui Kikai, Japan), and used for subsequent RNA extraction. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, USA), according to manufacturer’s instruction. The extracted RNA was treated with RNase-free DNase (TaKaRa, Japan), purified on a RNA-purification column (Qiagen, USA), and finally collected by ethanol precipitation. The RNA quality was evaluated on the Agilent Bioanalyzer 2100 (Agilent Technologies, USA), and RNA samples with RIN (RNA Integrity Number) value above 8 was used for subsequent cDNA synthesis.
To create cDNA sequencing library, mRNAs with poly(A) tail were isolated from the total RNA using beads with oligo(dT). Fragmentation buffer was then added to shear mRNAs into short fragments, which then served as templates for the synthesis of first strand of cDNA using random hexamer primers. cDNA library was then prepared using SureSelect Strand Specific RNA Library kit (Agilent Technologies, USA) according to the manufacturer’s specifications.
To create cDNA sequencing library, mRNAs with poly(A) tail were isolated from the total RNA using beads with oligo(dT). Fragmentation buffer was then added to shear mRNAs into short fragments, which then served as templates for the synthesis of first strand of cDNA using random hexamer primers. cDNA library was then prepared using SureSelect Strand Specific RNA Library kit (Agilent Technologies, USA) according to the manufacturer’s specifications.
5
Affinity Labeling of RNA Probes
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Sodium periodate (NaIO4), sodium cyanoborohydride (NaCNBH3), and lithium perchlorate (LiClO4) were purchased from Sigma-Aldrich. Alkylation was performed as previously described with some modifications (46 (link), 47 (link)). Briefly, freshly prepared 0.5 mL of 0.1 m NaIO4 was added to 10 μm CTBP1-AS RNA probe produced via in vitro transcription, and the mixture was incubated at 0°C for 20 min. The 3′-dialdehyde RNA was precipitated with 14 mL of 2% LiClO4 in acetone and then washed with 1 mL acetone. The pellet was dissolved in 5 μL of 0.1 m sodium acetate (pH 5.0) and then mixed with 0.25 mg Flag peptide. The reaction solution was mixed at room temperature for 3 h. The resulting imine moiety of the RNA–Flag was reduced by adding 10 μL of 1 m NaCNBH3 and then incubated at room temperature for 30 min. We obtained purified Flag-conjugated RNA using an RNA purification column (Qiagen, Hilden, Germany). The alkylation efficiency was estimated by measuring the amount of Flag–RNA recovered using NanoDrop. We used miRNA (miR-21; Thermo Fisher) to confirm the conjugation between RNA and Flag peptide using a gel shift assay. After the reaction, the reaction products were analyzed using denaturing urea polyacrylamide gel electrophoresis (15% acrylamide gel) with ethidium bromide staining.
6
RNA Isolation and cDNA Synthesis
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Total RNA was extracted from each plant tissue using the RNeasy Plant Mini Kit (Qiagen, USA) according to the manufacturer's protocol. The isolated RNA was treated with RNase-free DNase (Thermo Fisher Scientific, USA), followed by purification on RNA-purification column (Qiagen, USA), and finally collected by ethanol precipitation. RNA quality and quantity were then validated by Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA) and 1.5% agarose gel electrophoresis.
The mRNA fragments were used as templates to synthesize cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol.
The mRNA fragments were used as templates to synthesize cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol.
7
Aortic Transcriptome Analysis of Ldlr-/- Apobec1-/- Mice
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Aortae from the Ldlr-/-Apobec1-/- mice treated with nothing (CTL, N = 5), CLO (N = 5), or TIC (N = 5) were excised from the body and cleaned to remove adjacent connective and adipose tissue. We then isolated RNA individually from each aorta as described previously using RNA purification columns (Qiagen, Germantown, MD) [35 (link)]. We examined the quality of the RNA using the Agilent 2100 Bioanalyzer (Santa Clara, CA) and found that all samples had RNA integrity numbers higher than 8. We prepared the library using the Illumina (San Diego, CA) TrueSeq mRNA sample preparation kit per the manufacturer’s instructions. RNA sequencing was performed using the Illumina HISeq 1500 machine at the University of Texas Medical Branch NGS Core facility. The sequencing run yielded about 15 million reads on average. We aligned all reads using Tophat with the mm10 build of the mouse genome reference and annotation from the University of Southern California downloaded from Illumina’s iGenome website. We then examined transcript assembly and differential expression using Cufflinks with Refseq mRNAs [36 (link)]. Finally, we analyzed the RNA-seq data using the cummerbund package in R [37 (link)].
8
Rapid RNA Isolation from Surgical Samples
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The clinical samples were obtained directly from the OR (Operating Room) on ice. All samples were snap frozen in liquid nitrogen within 30′. The samples were then stored at −80°C and total RNA isolated with Qiagen RNA purification columns.
9
RT-qPCR Gene Expression Analysis
10
Quantifying Gene Expression in Tissues
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Total RNA (1 µg) isolated (Trizol Reagent, Invitrogen) from frozen tissues was converted to cDNA (cDNA reverse transcription kit, Invitrogen) and used to screen expression levels of the listed genes. Reactions were amplified in an ABI Prism 7500 FAST sequence detector (Applied Biosystems), and acquired data were analyzed using the ΔΔCt method to determine the expression level of each transcript normalized to the expression level of the housekeeping genes (Rplp0, Tbp, and/or Actb). RNAseq was conducted by Novogene, Co. Ltd. (Beijing, China) using total RNA DNase treated on RNA purification columns (QIAGEN).
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