Rna stabilization reagent

Manufactured by Qiagen

Sourced in Germany, United States, Canada

The RNA stabilization reagent is a solution designed to preserve the integrity of RNA samples. It quickly inactivates RNases and prevents degradation of RNA, ensuring the samples remain stable for downstream analysis.

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Lab products found in correlation

29 protocols using rna stabilization reagent

Extraction and Analysis of RNA from Embryonic Bones and Chondrocytes

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RNA was isolated directly from either E15.5 embryonic bones or primary
chondrocytes. For embryonic bones, E15.5 radius and ulna were dissected free from
connective tissue. The bones were then placed into RNA stabilization reagent (Qiagen,
Mississauga, ON, Canada) and passed through a series of syringes with 21G, 23G and 25G
needles (BD Biosciences, Mississauga, ON, Canada) to break the tissues. The RNA in the
homogenate was purified with RNeasy Micro Kit according to the manufacturer’s
instructions (Qiagen, Mississauga, ON, Canada). For primary chondrocytes, RNA was isolated
using TRIzol reagent, then reverse-transcribed by oligo (dT) and Superscript II reverse
transcriptase (Invitrogen, Burlington, ON, Canada). Quantitative real-time PCR was
performed using a Rotor-Gene 3000 instrument and data were analyzed using the Rotor-Gene
6.0.19 software (Montreal Biotech, Dorval, QC, Canada). Cyclophilin mRNA was used to
normalize gene expression. The primers (Supplementary Table 1) were purchased from Integrated DNA Technologies
(Coralville, IA, USA).

Li Z., Wu G., Sher R.B., Khavandgar Z., Hermansson M., Cox G.A., Doschak M.R., Murshed M., Beier F, & Vance D.E. (2014). Choline kinase beta is required for normal endochondral bone formation. Biochimica et biophysica acta, 1840(7), 2112-2122.

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Profiling Hair Follicle and Basal Cell Carcinoma Transcriptomes

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All the samples were provided through the Department of Surgery and the Department of Dermatology and Skin Science, University of British Columbia, with approval from the University Clinical Research Ethics Board. Samples of human HFs were collected from scalp biopsies of normal individuals undergoing cosmetic procedures, while nodular BCCs and normal skin were obtained from patients undergoing surgical resection. All the nodular BCC samples and normal skin epithelium were taken from the facial area of donors. Only tissue from patients that had not been treated with preoperative chemotherapy or other therapeutic approaches was selected for analysis. BCC morphological subtypes were described and clinically classified during Mohs surgery and initial diagnoses were subsequently confirmed by formalin-fixed, paraffin-embedded histological assessment of the tumors.
Hair follicles (n=10-20/subject) were microdissected to remove the sebaceous gland and upper HF infundibulum and the lower one third, including the hair bulb. The dermal sheath was also removed, leaving the inner and outer root sheaths, including the bulge region, for analysis. Normal skin samples were microdissected to isolate skin epithelium from the dermal component. Samples collected for microarray/qPCR were immediately stored in an RNA stabilization reagent (Qiagen Inc., Toronto, ON, Canada).

Shi F.T., Yu M., Zloty D., Bell R.H., Wang E., Akhoundsadegh N., Leung G., Haegert A., Carr N., Shapiro J, & McElwee K.J. (2017). Notch signaling is significantly suppressed in basal cell carcinomas and activation induces basal cell carcinoma cell apoptosis. Molecular Medicine Reports, 15(4), 1441-1454.

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West Nile Virus Infection in SPF Chickens

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In independent experiments, SPF chickens were inoculated subcutaneously (100 µL inoculum/chick) in the cervical region at the age of 1 day with a dose of 10³ TCID₅₀ of West Nile virus diluted in sterile PBS. The dose of 10³ TCID₅₀ was chosen because it is close to the dose usually inoculated by C. pipiens mosquitoes during natural infections (i.e. 10^4.3 PFU, [39 (link)]), and because we showed in a previous work [37 (link)] that this dose allows a significant discrimination between different pathotypes of lineage 1 West Nile virus strains in the SPF chicken model, based on mortality rates. The number of inoculated chicks amounted to 52 for Hun2004, 51 for Aus2008 and 42 for Gr2011. On 2, 5, 7, 9, 12 and 14 days post-infection (dpi), 5 chickens per sub-group were sacrificed by exsanguination. Blood was immediately collected in microtubes and allowed to clot for 6 h at room temperature (RT°). After centrifugation at 2345 g for 5 min, serum was collected and aliquots stored at −20 or −80 °C for further determination of neutralizing antibodies titers or RNA extraction, respectively. Two to three wing primary feathers were sampled from every sacrificed bird and stored in 600 µL of RNA later (RNA Stabilization Reagent, Qiagen Benelux B.V., The Netherlands) at −80 °C for further RNA extraction.

Dridi M., Van Den Berg T., Lecollinet S, & Lambrecht B. (2015). Evaluation of the pathogenicity of West Nile virus (WNV) lineage 2 strains in a SPF chicken model of infection: NS3-249Pro mutation is neither sufficient nor necessary for conferring virulence. Veterinary Research, 46, 130.

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Quantitative Analysis of Sestrin Expression

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For quantitative real-time PCR, six to seven fresh dissected OC, stria vascularis, modiolus and brain explants were used for each genotype. In the case of treated samples, six OC explants without the apex region were used for each condition and each genotype. Dissected organs were stored in RNA stabilization reagent (Qiagen, Hombrechtikon, Switzerland) at −20 °C until needed. Total RNA was extracted using an RNeasy Plus Mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were determined using the Nanodrop ND-1000 (Thermo Fisher Scientific). cDNA synthesis was carried out using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific). The TaqMan gene expression assays for mouse Sesn1 (Mm01185732_m1), Sesn2 (Mm00460679_m1), Sesn3 (Mm01171504_m1) and GAPDH (Mm99999915_g1) were purchased from Applied Biosystems. Quantitative PCR was performed using TaqMan gene expression assays and the TaqMan Fast Advanced Master Mix kit (Applied Biosystems) on an ABI 7500 Fast Real-Time PCR instrument (Applied Biosystems). Each reaction was performed in triplicate. Relative mRNA levels were determined using the 2−ΔΔCT method and normalized to the housekeeping gene GAPDH.

Ebnoether E., Ramseier A., Cortada M., Bodmer D, & Levano-Huaman S. (2017). Sesn2 gene ablation enhances susceptibility to gentamicin-induced hair cell death via modulation of AMPK/mTOR signaling. Cell Death Discovery, 3, 17024-.

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Lipid Profile and Liver ACE Expression

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Blood samples were assembled through retroorbital route twice; once in the beginning of the study after fasting for overnight and the other in the end of nine weeks before sacrificing animal. Serum samples were centrifuged (15 min, 3000 xg) and kept at −80 °C to screen lipid profile, glucose homeostasis and liver enzymes. At the last step, rats’ livers were carefully dissected out and saved at –80 °C in RNA later solution (RNA stabilization reagent- QIAGEN) to estimate the expression levels of ACE1 and ACE2 by Quantitative Polymerase Chain Reaction (qPCR).

Mohater S., Qahtan S., Alrefaie Z, & Alahmadi A. (2023). Vitamin D improves hepatic alterations in ACE1 and ACE2 expression in experimentally induced metabolic syndrome. Saudi Pharmaceutical Journal : SPJ, 31(9), 101709.

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RNA Extraction and qPCR Analysis

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Nerve tissue samples were placed in RNA Stabilization Reagent (Qiagen, Valencia, CA, USA). The total RNA fraction was extracted using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Reverse transcription using a reverse transcription kit (PrimeScript™ RT reagent Kit, TaKaRa, Shiga, Japan), and the resulting cDNA was subjected to real-time fluorescent quantitative PCR amplification. The relative expression values of each mRNA were calculated using comparative CT analysis and were normalized using glyceraldehyde phosphate dehydrogenase (Gapdh) mRNA for each data point. All reactions were run in triplicate. The primers used in RT-PCR assays are shown in Table 1.

Lin Y.F., Xie Z., Zhou J., Yin G, & Lin H.D. (2019). Differential gene and protein expression between rat tibial nerve and common peroneal nerve during Wallerian degeneration. Neural Regeneration Research, 14(12), 2183-2191.

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Quantitative RT-PCR Analysis of ERβ Expression

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Total RNA was isolated from tissues that were stored in RNA stabilization reagent (Qiagen, Valencia, CA) using TRIzol reagent (Invitrogen, Carlsbad, CA) after a quick liquid nitrogen grind. The quality and quantity of RNA were determined from its optical density in an ethidium bromide-stained 1% agarose gel, and 2 µg of RNA was subsequently used to generate cDNA with the M-MLV reverse transcription kit (Invitrogen, Carlsbad, CA). Real-time quantitative PCR was performed with a LightCycler 480 System (Roche Diagnostics GmbH, Mannheim, Germany). The LightCycler 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany) assay was used for total ERβ amplification and GAPDH was used as a reference gene. The 20 µl reaction system contained 10 µl of SybGreen mix, 10 pmol of each primer, and 50–100 ng of cDNA template. The primers used are listed in Table II. Relative expression levels of total ERβ were calculated by N = 2^–_Δ^Ct(GAPDH^–^ERβ) [18 (link)], where N is the relative quantity of mRNA expression.

Gao L., Qi X., Hu K., Zhu R., Xu W., Sun S., Zhang L., Yang X., Hua B, & Liu G. (2016). Estrogen receptor β promoter methylation: a potential indicator of malignant changes in breast cancer. Archives of Medical Science : AMS, 12(1), 129-136.

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Breast Cancer Tissue Collection and Analysis

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The study protocol was approved by the Institutional Review Board and all participants signed a written consent form. Tissues of 132 breast cancer objects were collected from September 2010 to October 2011 at the Anyang Cancer Hospital in the Henan province of China. All participants were Chinese Han women, mean age 50.91 ±9.88 years, and baseline characteristics are shown in Table I. None of them had received preoperative hormonal therapy. Cancerous and adjacent normal tissues were collected from each patient during surgery. Normal breast tissues were obtained at least 1 cm distant to the edge of cancerous breast tissue. Subsamples of tissues were removed prior to DNA extraction for conventional fixed, embedded and H + E staining. Histopathological categories including benign and malignant characterizations were verified by two experienced pathologists. Each tissue was divided into two parts, and one was placed in RNA Stabilization Reagent (Qiagen, Valencia, CA), while the other was stored at –80°C until extraction.

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Rat Splenic Tissue RNA Extraction

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Splenic tissue from rats (~30 mg) was stored in RNA later RNA Stabilization Reagent (Qiagen) for 24 hours at 4°C, and thereafter stored at -80°C until extraction. Total RNA extraction was performed using RNEasy Mini kit (Qiagen) and a FastPrep-24 Homogenizer. The kit included an on-column DNA digestion step. cDNA was synthesized from 1 μg RNA using SuperScript III First Strand Synthesis Supermix kit (ThermoFisher Scientific). qPCR was performed using SYBR Green Master Mix (ThermoFisher Scientific) and a Quant Studio 5 PCR system. Primer sequences used are listed in S3 Table. In all experiments Gapdh was used as a reference gene.

Steiner S.E., Choong F.X., Antypas H., Morado-Urbina C.E., Schulz A., Bersellini Farinotti A., Bas D.B., Svensson C.I., Richter-Dahlfors A, & Melican K. (2021). UPEC kidney infection triggers neuro-immune communication leading to modulation of local renal inflammation by splenic IFNγ. PLoS Pathogens, 17(5), e1009553.

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Quantification of Rabbit mRNA Expression

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Rabbit tissues were incubated in RNA later (RNA stabilization reagent, Qiagen) and mechanically disrupted before extraction of total RNA using RNeasy^® lipid tissue mini kit (Qiagen) according to the manufacturer’s protocol. Corresponding cDNAs were synthetized using QuantiTect reverse transcription kit (Qiagen). Quantification was performed by TaqMan using Takyon Rox probe mastermix dTTP blue (Eurogentec). Sequences of specific primers and probes used are detailed in Supplementary Table 1. Cycling conditions were as follows: first step of denaturation at 95° C during 3 min., followed by 40 cycles of amplification (95° C 15 sec, 58° C 15 sec and 68° C 15 sec). Fluorescence was measured during the 68° C step incubation using a Realplex² Mastercycler (Eppendorf). The specificity of the PCR products was verified by sequencing. Messenger RNA expression levels were normalized based on the EF1α reporter gene.

Laude H.C., Caval V., Bouzidi M.S., Li X., Jamet F., Henry M., Suspène R., Wain-Hobson S, & Vartanian J.P. (2018). The rabbit as an orthologous small animal model for APOBEC3A oncogenesis. Oncotarget, 9(45), 27809-27822.

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