Rnaprotect

Manufactured by Qiagen

Sourced in Germany, United States, United Kingdom, Italy, Spain, Netherlands

RNAprotect is a reagent designed to immediately stabilize and protect RNA in samples. It helps prevent RNA degradation and changes in gene expression during sample collection, storage, and transportation.

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Lab products found in correlation

Close spelling variants of this product

RNAprotect Bacteria Reagent (429 citations) Cell RNAProtect (2 citations) Bacterial RNAprotect (6 citations) RNAprotect Bacteria Reagent and RNeasy Mini Kit (2 citations) RNAprotect Animal Blood Tubes (33 citations) RNAprotect Bacteria Reagent kit (3 citations) RNAprotect Bacteria (10 citations) RNAprotect Saliva Reagent (4 citations) RNAprotect Tissue Reagent (38 citations) RNAprotect Cell Mini Kit (2 citations)

311 protocols using rnaprotect

Monitoring Microbial RNA Dynamics

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Approximately every 12 h (at each low tide) for three consecutive days duplicate samples were removed from each of three chambers using sterile syringes prefilled with RNAprotect (Qiagen, Valencia, CA) at a 2:1 ratio of RNAprotect to cell culture, following the manufacturer's protocol. A previously optimized RNA extraction assay was then performed as described by Williams et al. [2014a]. RNA quality and quantity was assessed using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE), and samples having a 260/280 nm ratio of ≥1.7 and a concentration of ≥75 ng/μL were stored at −80°C. Using endpoint PCR, determination of DNA contamination was performed as previously described [Phippen and Oliver, 2015b; Williams et al., 2014a] utilizing the species‐specific gene target, vvhA.

Phippen B.L, & Oliver J.D. (2017). Impact of hypoxia on gene expression patterns by the human pathogen, Vibrio vulnificus, and bacterial community composition in a North Carolina estuary. GeoHealth, 1(1), 37-50.

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RNA Extraction from Blood and Tissue Samples

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On procedure days, 100 μL of blood from K2-EDTA collection tubes was collected prior to centrifugation and was added to 600 μL of AVL viral lysis buffer with 6 μL carrier RNA (Qiagen, 52906) for RNA extraction. For tissues, approximately 100 mg was stored in 1 mL RNAprotect (Qiagen, 1018087) for at least 4 days for stabilization. RNAprotect was completely removed, and tissues were homogenized in 600 μL RLT buffer (Qiagen, 74004) and 1% β-mercaptoethanol in a 2 mL cryovial using a TissueLyser II (Qiagen, 85300) and 0.2 mm ceramic beads. The tissues sampled included axillary and inguinal lymph nodes, liver, spleen, kidney, adrenal gland, lung, pancreas, urinary bladder, ovary or testis, and eye. All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the biosafety level 4 (BSL-4) laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen, 52906) and from tissues using the RNeasy Mini kit (Qiagen, 74004) according to the manufacturer’s instructions supplied with each kit.

Cross R.W., Bornholdt Z.A., Prasad A.N., Woolsey C., Borisevich V., Agans K.N., Deer D.J., Abelson D.M., Kim D.H., Shestowsky W.S., Campbell L.A., Bunyan E., Geisbert J.B., Dobias N.S., Fenton K.A., Porter D.P., Zeitlin L, & Geisbert T.W. (2022). Combination therapy with remdesivir and monoclonal antibodies protects nonhuman primates against advanced Sudan virus disease. JCI Insight, 7(10), e159090.

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Hypothalamic Microdissection for RNA Profiling

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All mice were sacrificed at ZT 5. Brains were immediately extracted and dropped into ice cold Hanks’ Balanced Salt Solution (HBSS). After 2 minutes, brains were embedded in low melting point agarose (Precisionary Instruments, Natick, MA) and sectioned at 400 μm on a Compresstome VF-200 Vibrating Microtome (Precisionary Instruments, Natick, MA, USA) into DNAse/RNAse free 1x PBS. Hypothalamic sections of interest were immediately collected into RNAprotect (Qiagen, Hilden, Germany) and kept in RNAprotect at 4°C overnight. The next day Lepr-Cre; TdTomato positive cells were visualized using a fluorescent stereoscope (Leica, Wetzlar, Germany). tdTomato fluorescence was used to approximate DMH and SCN boundaries during microdissection of these regions. DMH and SCN microdissected tissue samples were placed into Eppendorf tubes separated by brain region and feeding condition, and stored in −80°C until nuclei isolation.

Tang Q., Godschall E., Brennan C.D., Zhang Q., Abraham-Fan R.J., Williams S.P., Güngül T.B., Onoharigho R., Buyukaksakal A., Salinas R., Olivieri J.J., Deppmann C.D., Campbell J.N., Podyma B, & Güler A.D. (2023). A leptin-responsive hypothalamic circuit inputs to the circadian feeding network. bioRxiv.

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Transcriptional Response to Copper Stress

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For the microarray experiments, S. pyogenes HSC5, revertant, and ΔcopA strains were grown in 30 mL of ThyB at 37 °C. At early exponential phase growth (OD600 = 0.3) the strains were exposed to a shock treatment of 100 μM CuSO₄ for 30 min. The treated cultures were then diluted into RNA Protect (Qiagen) at a ratio of 1:2 (culture:RNA Protect) and harvested by centrifugation at 5000 ×g for 10 min. Total RNA was extracted using a RiboPure RNA Purification kit (ThermoFisher) according to the manufacturer’s protocols. The concentration and purity of extracted RNA for each sample were determined by the absorbance values ratio at 260/280 nm.

Wang J., Chun H.J., Wong W., Spencer D.M, & Lenardo M.J. (2001). Caspase-10 is an initiator caspase in death receptor signaling. Proceedings of the National Academy of Sciences of the United States of America, 98(24), 13884-13888.

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Transcriptional Response to Copper Stress

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Dao T.H., Iverson A., Neville S.L., Johnson M.D., McDevitt C.A, & Rosch J.W. (2023). The role of CopA in Streptococcus pyogenes copper homeostasis and virulence. Journal of inorganic biochemistry, 240, 112122.

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RNA-seq Protocol for Bacterial Transcriptome Analysis

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Ten milliliters of bacterial culture was collected from triplicate cultures growing exponentially in minimal medium containing glucose (OD at 600 nm [OD₆₀₀] = 0.45 to 0.5) or 10 min after centrifugation and resuspension in minimal medium lacking a carbon source. Cell pellets were immediately frozen on dry ice and stored at −80°C. The mRNA was stabilized by treatment with 10 ml of RNAprotect (Qiagen) (diluted 2 parts RNAprotect to 1 part water) prior to extraction using the RNeasy minikit (Qiagen) with on-column DNase I treatment. The eluates were treated with Turbo DNase (Invitrogen) for 30 min before a subsequent round of purification using the RNeasy minikit. RNA-seq was performed at the Yale Center for Genome Analysis, where the RNA quality was examined using a BioAnalyzer (Agilent) before rRNA was depleted using the RiboZero bacterial kit (Illumina). Approximately 50 million 75-bp paired-end reads per sample were collected using a HiSeq 4000 (Illumina).

Townsend GE I.I., Han W., Schwalm ND I.I.I., Hong X., Bencivenga-Barry N.A., Goodman A.L, & Groisman E.A. (2020). A Master Regulator of Bacteroides thetaiotaomicron Gut Colonization Controls Carbohydrate Utilization and an Alternative Protein Synthesis Factor. mBio, 11(1), e03221-19.

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Gametocyte Enrichment for Molecular Detection

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In a subset of experiments from Burkina Faso, the parasite population was enriched for gametocytes prior to molecular parasite detection. This was performed using MACS LS columns (Miltenyi Biotec, UK) as previously published (Karl et al., 2011 (link)) with minor modifications. Immediately after venipuncture, 500 µl of whole blood were re-suspended in 5 ml of magnetic fractionation buffer (1× PBS pH7.4, 0.5% BSA, 2 mM EDTA) and passed in batches over the LS column. The flow-through, containing asexual parasites, was collected and briefly centrifuged, and the supernatant was removed and the cell pellet re-suspended in 500 µl of RNA protect (Qiagen). The gametocyte fraction was eluted, briefly centrifuged and suspended in 5× RNA protect (Qiagen). All steps were performed at 37 °C.

Grignard L., Gonçalves B.P., Early A.M., Daniels R.F., Tiono A.B., Guelbéogo W.M., Ouédraogo A., van Veen E.M., Lanke K., Diarra A., Nebie I., Sirima S.B., Targett G.A., Volkman S.K., Neafsey D.E., Wirth D.F., Bousema T, & Drakeley C. (2018). Transmission of molecularly undetectable circulating parasite clones leads to high infection complexity in mosquitoes post feeding. International Journal for Parasitology, 48(8), 671-677.

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Modulating Cell Signaling Pathways

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For TBK1/IKKε inhibition, cells were plated for mono-culture, direct or indirect co-culture in media containing either DMSO or drugs. TBK1/IKKε inhibitor (250nM) compounds were kindly provided by GlaxoSmithKline (GSK); GSK2 (GSK2286574A) and GSK3 control (GSK2282513A). The same method was used for perturbing IRF3 nuclear location using Dynasore (50μM) (Selleckchem #S8047). Cells were fixed after 6 hours before immunofluorescent staining. Again, the same method was used for Gap junction inhibition, using Tonabersat 40 μM (Sigma SML1354) and Enoxolone 100 μM (Abcam ab142579). Cells were cultured for 20 hours before harvesting in RNA Protect (Qiagen #76526) for gene expression analyses. AZD6738 (Selleckchem #S7693) was used at 1μM to treat the cancer cells for 48h, after that cells were trypsinized and plated in direct or indirect co-culture with fibroblasts or on their own for 20 hours before harvesting in RNA Protect (Qiagen #76526) for gene expression analyses. For micronuclei analysis, after treatment cells were fixed with 4% paraformaldehyde and stained with DAPI 1:1000 (Sigma D9542) for imaging in Zeiss LSM510 microscope. Images were analysed using ImageJ.

Arwert E.N., Milford E.L., Rullan A., Derzsi S., Hooper S., Kato T., Mansfield D., Melcher A., Harrington K.J, & Sahai E. (2020). STING and IRF3 function in stromal fibroblasts enables sensing of genomic stress in cancer cells thereby undermining oncolytic viral therapy. Nature cell biology, 22(7), 758-766.

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RNA Extraction from Cells on Devices

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RNA was extracted from devices immediately after the flow was stopped and the media removed, by adding RNAprotect (QIAGEN, Valencia, CA, United States) directly to the cells and scraping the gelatin layer with a pipette tip to ensure that all the cells were detached. The RNAprotect containing the cells was then collected and the RNeasy Mini Kit (QIAGEN, Valencia, CA, United States) was used as detailed in the Supplementary Material. Since sample contamination with gelatin present in the wells clogged the spin columns provided in the RNeasy kit, alternatively, samples were collected by adding TRIzol Reagent (ThermoFisher Scientific, Wilmington, DE, United States) directly to the cells, and RNA was isolated as detailed in the Supplementary Material. The use of TRIzol allowed high-quality RNA to be extracted at high yield (Alves et al., 2016 (link)). RNA integrity and purity were then verified using the Agilent RNA 6000 Nano Kit, as described in the Supplementary Material (Agilent Technologies, Santa Clara, CA, United States).

Trevisan B., Morsi A., Aleman J., Rodriguez M., Shields J., Meares D., Farland A.M., Doering C.B., Spencer H.T., Atala A., Skardal A., Porada C.D, & Almeida-Porada G. (2021). Effects of Shear Stress on Production of FVIII and vWF in a Cell-Based Therapeutic for Hemophilia A. Frontiers in Bioengineering and Biotechnology, 9, 639070.

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Brain Dissection for Single-Cell Analysis

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All mice were euthanized at ZT5. Brains were immediately extracted and dropped into ice-cold Hanks’ balanced salt solution. After 2 min, brains were embedded in low melting point agarose (Precisionary Instruments, Natick, MA) and sectioned at 400 μm on a Compresstome VF-200 Vibrating Microtome (Precisionary Instruments, Natick, MA, USA) into deoxyribonuclease/ribonuclease-free 1× phosphate-buffered saline (PBS). Hypothalamic sections of interest were immediately collected into RNAprotect (QIAGEN, Hilden, Germany) and kept in RNAprotect at 4°C overnight. The next day Lepr-Cre;TdTomato–positive cells were visualized using a fluorescent stereoscope (Leica, Wetzlar, Germany). tdTomato fluorescence was used to approximate DMH and SCN boundaries during microdissection of these regions. DMH and SCN microdissected tissue samples were placed into Eppendorf tubes separated by brain region and feeding condition and stored in −80°C until nucleus isolation.

Tang Q., Godschall E., Brennan C.D., Zhang Q., Abraham-Fan R.J., Williams S.P., Güngül T.B., Onoharigho R., Buyukaksakal A., Salinas R., Sajonia I.R., Olivieri J.J., Calhan O.Y., Deppmann C.D., Campbell J.N., Podyma B, & Güler A.D. (2023). Leptin receptor neurons in the dorsomedial hypothalamus input to the circadian feeding network. Science Advances, 9(34), eadh9570.

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