Paxgene blood rna kit

Manufactured by Preanalytix

Sourced in Switzerland, Germany, United States

The PAXgene Blood RNA Kit is a laboratory equipment product designed for the collection, stabilization, and purification of RNA from whole blood samples. It provides a standardized method for the preservation of RNA integrity and yield.

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171 protocols using paxgene blood rna kit

Optimizing RNA Extraction from EDTA Blood

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For RNA extraction from original Paxgene tubes, samples were thawed on ice for at least 2 h and PAXgene Blood RNA Kit (PreAnalytix, Hombrechtikon, Switzerland) was used to extract blood RNA according to the manufacturer’s manual. This method is referred to as Paxgene Control or PP. For EDTA tubes, we had two strategies to optimise the RNA quantity and quality. The first was to replicate and optimise a reference protocol based on transferring EDTA blood to PAXgene tubes before extraction. Several conditions were then tested including (1) Different extraction kits: PAXgene Blood RNA Kit (PreAnalytix, Hombrechtikon, Switzerland) versus MagMAX blood RNA Isolation Kit for PAXgene (Thermofisher Scientific, MA, USA), (2) different post-transfer incubation time: 2 h versus overnight, and (3) same day extraction vs refreezing the samples for later extraction. The second strategy was to optimise the Nucleospin blood RNA extraction kit (Macherey–Nagel, Westfalen, Germany), which was primarily meant to be used for fresh blood but has been shown to produce a much higher yield of RNA compared to other protocols using frozen blood^{15 (link),16 (link)}.

Nguyen L.T., Pollock C.A, & Saad S. (2024). Extraction of high quality and high yield RNA from frozen EDTA blood. Scientific Reports, 14, 8628.

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Blood Collection and RNA Extraction

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2.5 ml whole blood was collected at the time of recruitment (before or within 24 h of commencing TB treatment in suspected patients) in PAXgene blood RNA tubes (PreAnalytiX), frozen within 3 h of collection, and later extracted using PAXgene blood RNA kits (PreAnalytiX). RNA was shipped frozen and stored at −80°C.

Gliddon H.D., Kaforou M., Alikian M., Habgood-Coote D., Zhou C., Oni T., Anderson S.T., Brent A.J., Crampin A.C., Eley B., Heyderman R., Kern F., Langford P.R., Ottenhoff T.H., Hibberd M.L., French N., Wright V.J., Dockrell H.M., Coin L.J., Wilkinson R.J, & Levin M. (2021). Identification of Reduced Host Transcriptomic Signatures for Tuberculosis Disease and Digital PCR-Based Validation and Quantification. Frontiers in Immunology, 12, 637164.

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Quantifying mRNA Expression of Glucocorticoid Receptor and β2-Adrenergic Receptor

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We collected 2.5mL of peripheral blood into PAXgene Blood RNA tubes in a randomly selected subsample of 37 participants (for cost purposes) to measure expression of the α isomer of the glucocorticoid receptor (GR) and β2-adrenergic receptor (β2-AR) mRNA by white blood cells using real-time RT-PCR. Total RNA was extracted using PAXgene Blood RNA Kits (Pre-Analytix, Hombrechtikon, Switzerland). PCRs were conducted on an Applied Biosystems Prism 7000 Sequence Detection System (Foster City, CA) using commercially available one-step assays (TaqMan Gene Expression Assay developed in partnership with Applied Biosystems and based on RefSeq NM_000176 for GRα; TaqMan Gene Expression Assay #Hs00240532 for β2-AR; Applied Biosystems). Using the delta CT method, values of each target mRNA were adjusted for expression of a housekeeping gene, 18S, measured in parallel (TaqMan Gene Expression Assay #Hs99999901; Applied Biosystems). Results are expressed as relative quantities of mRNA, such that higher values indicate greater expression of the target mRNA relative to the sample range. Because PCR yields data on a log-2 base scale, each unit difference in relative quantity indicates a twofold difference in expression.

Ehrlich K.B., Miller G.E, & Chen E. (2015). Harsh Parent-Child Conflict is Associated with Decreased Anti-Inflammatory Gene Expression and Increased Symptom Severity in Children with Asthma. Development and psychopathology, 27(4 Pt 2), 1547-1554.

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Real-Time qPCR Quantification of RDH12 and PGK1

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RNA was extracted from peripheral blood using PAXgene Blood RNA Kits (PreAnalytix). Coding DNA was made with 250 ng total RNA using SuperScript II reverse transcriptase (RT) (Invitrogen), and multiplexed RT-coupled polymerase chain reaction (PCR) was run the same day in triplicate using Taqman probes with conjugated FAM for RDH12 amplification and conjugated VIC for PGK1 amplification (Applied Biosystems). Taqman probes were designed by Thermo Fisher Scientific, assay ID Hs00288401_m1 using Refseq NM_152443.2 for RDH12 and assay ID Hs00943178_g1 using NM_000291.3 for PGK1. PCR reactions were run in the Biorad iCycler. Relative RDH12 transcript levels were normalized to PGK1.

Fahim A.T., Bouzia Z., Branham K.H., Kumaran N., Vargas M.E., Feathers K.L., Perera N.D., Young K., Khan N.W., Heckenlively J.R., Webster A.R., Pennesi M.E., Ali R.R., Thompson D.A, & Michaelides M. (2019). Detailed Clinical Characterisation, Unique Features, and Natural History of Autosomal Recessive RDH12-Associated Retinal Degeneration. The British journal of ophthalmology, 103(12), 1789-1796.

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Quantitative Real-Time PCR for Gene Expression Analysis

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RNA was isolated using PAXgene blood RNA kits (PreAnalytiX GmbH), and quantified using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware). Reverse transcription of total RNA was carried out using iScriptII Reverse Transcription Supermix (Bio-Rad Laboratories, Mississauga, Canada). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed by adding 1 µL cDNA and 0.5 mM primers (Table 1) to SsoFast EvaGreen (Bio-Rad). PCR amplification was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad). Melt curves were analyzed to confirm that non-specific products were absent. The fluorescence detection threshold was set above the non-template control background within the linear phases of PCR amplifications and the cycle threshold (Ct) of each reaction was detected. Gene expression was analyzed using the ΔΔCt method and results are presented as fold-differences normalized to housekeeping gene (β-actin).

Labonté L., Coulombe P., Zago M., Bourbeau J, & Baglole C.J. (2014). Alterations in the Expression of the NF-κB Family Member RelB as a Novel Marker of Cardiovascular Outcomes during Acute Exacerbations of Chronic Obstructive Pulmonary Disease. PLoS ONE, 9(11), e112965.

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Blood RNA Extraction Using PAXgene

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Whole blood was collected (2.5 mL) in PAXgene blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland), incubated for 2 h at ambient temperature, frozen at –20 °C within 24 h of collection, and stored at –80 °C. Total RNA was extracted using PAXgene blood RNA kits (PreAnalytiX) according to the manufacturer’s instructions.

Kuiper R., Wright V.J., Habgood-Coote D., Shimizu C., Huigh D., Tremoulet A.H., van Keulen D., Hoggart C.J., Rodriguez-Manzano J., Herberg J.A., Kaforou M., Tempel D., Burns J.C, & Levin M. (2022). Bridging a diagnostic Kawasaki disease classifier from a microarray platform to a qRT-PCR assay. Pediatric Research, 93(3), 559-569.

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Glucocorticoid and β2-Adrenergic Receptor mRNA Expression

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At visits two through five, 2.5 mL of whole blood was drawn into PAXgene Blood RNA tubes through antecubital venipuncture to measure the expression of messenger RNA (mRNA) for the α isoform of the Glucocorticoid Receptor (GR) and for β₂-Adrenergic Receptor (β₂-AR). Total RNA was extracted using PAXgene Blood RNA Kits (Pre-Analytix, Hombrechtikon, Switzerland) and real-time reverse transcription-polymerase chain reaction was executed using commercially-available one-step assays (TaqMan Gene Expression Assay developed in partnership with Applied Biosystems and based on RefSeq NM_000176 for GR-α; TaqMan Gene Expression Assay #Hs00240532 for β₂-AR; Applied Biosystems). Values of each target mRNA were adjusted for expression of a housekeeping gene, 18S, using the delta CT method. Results are expressed as relative quantities of mRNA on a log 2 base scale, where higher values indicate greater expression of the target mRNA and each unit difference in relative quantity indicates a twofold difference in expression.

Manczak E.M., Dougherty B, & Chen E. (2019). Parental Depressive Symptoms Potentiate the Effect of Youth Negative Mood Symptoms on Gene Expression in Children with Asthma. Journal of abnormal child psychology, 47(1), 99-108.

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Biologic Agent Blood Sample Collection

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Before administration of a biologic agent, blood samples were collected in PAXgene Blood RNA tubes [14 (link)] (PreAnalytiX, Hombrechtikon, Switzerland). Total RNAs were extracted using PAXgene Blood RNA kits (PreAnalytiX) following the manufacturer’s instructions. Total RNA quantity and quality were determined using a NanoDrop 1000 spectrophotometer (NanoDrop Products/Thermo Fisher Scientific, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All RNA samples fulfilled both of the following criteria: RNA integrity >6.5 and optical density at 260/280 nm >1.6.

Nakamura S., Suzuki K., Iijima H., Hata Y., Lim C.R., Ishizawa Y., Kameda H., Amano K., Matsubara K., Matoba R, & Takeuchi T. (2016). Identification of baseline gene expression signatures predicting therapeutic responses to three biologic agents in rheumatoid arthritis: a retrospective observational study. Arthritis Research & Therapy, 18, 159.

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Blood RNA extraction and analysis

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Blood from the patients was collected in PAXgene Blood RNA tubes (PreAnalytiX) at baseline and at 6 months after the initiation of ABA treatment. Total RNAs were extracted using PAXgene Blood RNA Kits (PreAnalytiX) following the manufacturer’s instructions. The total RNA quantity and quality were determined using a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies).

Yokoyama-Kokuryo W., Yamazaki H., Takeuchi T., Amano K., Kikuchi J., Kondo T., Nakamura S., Sakai R., Hirano F., Nanki T., Koike R, & Harigai M. (2020). Identification of molecules associated with response to abatacept in patients with rheumatoid arthritis. Arthritis Research & Therapy, 22, 46.

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RNA Preservation and Analysis Protocol

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Total RNA was extracted from PAXgene RNA blood tubes using the PAXgene Blood RNA Kit (PreAnalytix, Hilden, Germany) according to the manufacturer's protocol using only RNase-free equipment and solutions. The RNA was eluted in 80 µl of nuclease free H₂O. RNA yield was quantified using a NanoDrop 1000 spectrophotometer. Each isolated RNA sample was divided into 12 aliquots of 6 µl each, half of which were pipetted into 1.5 ml Eppendorf tubes and immediately stored at −80°C. The remaining six samples isolated from each blood sample were desiccated in Biomatrica RNAstable 1.5 ml tubes by room temperature vacuum centrifugation using an Eppendorf Speed Vacuum Concentrator (Eppendorf, Hamburg, Germany). The desiccated RNA aliquots were then stored in a special humidity controlled dry storage cabinet. Paired aliquots of each sample (frozen and desiccated) were analyzed every two months (except at 10 months), RNAstable tubes were rehydrated in 6 µl of nuclease-free H₂O, and the RNA quality was analyzed using an Agilent 2100 bioanalyzer to obtain RINs and by quantitative real time PCR at two, six, and 12 months (Fig. S3).

Seelenfreund E., Robinson W.A., Amato C.M., Tan A.C., Kim J, & Robinson S.E. (2014). Long Term Storage of Dry versus Frozen RNA for Next Generation Molecular Studies. PLoS ONE, 9(11), e111827.

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