Rneasy isolation kit

Manufactured by Qiagen

Sourced in Germany, United States, Spain, Switzerland

The RNeasy isolation kit is a laboratory product designed to isolate and purify RNA from various biological samples. It utilizes a silica-based membrane technology to efficiently capture and wash RNA, allowing for high-quality RNA extraction.

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201 protocols using rneasy isolation kit

Quantitative Real-Time PCR Analysis

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Total RNA was isolated using TRIzol reagent (Invitrogen) and purified using the RNeasy isolation kit (Qiagen) according to the manufacturer’s protocol. 1 μg of total RNA was reverse transcribed using iScript RT Supermix (Bio-Rad #1708841), following the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) analysis was performed in duplicate using iQ SYBR green Supermix (Bio-Rad #1708880) on an iCycler Real-Time Detection System (Bio-Rad). The mRNA level was normalized to GAPDH or 18S as a housekeeping gene. For mouse tissues, total liver RNA from C57BL6 and Apobec1^−/−;Ldlr^–/+ mice was isolated using the Bullet Blender Tissue Homogenizer (Next Advance) in TRIzol and purified using the RNeasy isolation kit (Qiagen). 1 μg of total RNA was reverse transcribed and gene expression assessed as above. Primer sequences are listed in Supplementary Table 1.

Goedeke L., Canfrán-Duque A., Rotllan N., Chaube B., Thompson B.M., Lee R.G., Cline G.W., McDonald J.G., Shulman G.I., Lasunción M.A., Suárez Y, & Fernández-Hernando C. (2021). MMAB promotes negative feedback control of cholesterol homeostasis. Nature Communications, 12, 6448.

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Cytokine Expression Analysis by qRT-PCR

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The relative expression of pro-inflammatory cytokines (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and anti-inflammatory cytokine (IL-10)) were determined by qRT-PCR. Tissue was homogenized in TRIzol reagent (Life Technologies, Carlsbad, CA, USA), and total ribonucleic acid (total RNA) was extracted using the RNeasy isolation kit (Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. cDNA was synthesized from 200 ng total RNA using M-MLV transcriptase (Life Technologies, Carlsbad, CA, USA). Next, real-time PCR reaction was performed on a LightCycler 96W (Roche, Vilvoorde, Belgium) with Taqman Fast Universal PCR Master Mix and Taqman gene expression assays (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) (IL-1β (Rn00580432_m1), TNF-α (Rn00562055), and IL-10 (Rn00563409)). A three-step amplification program was used: 95 °C for 10 min, followed by 45 cycles of amplification (95 °C for 10 s, 60 °C for 15 s, 72 °C for 10 s). Target messenger RNA (mRNA) expression for cytokines was quantified relative to the housekeeping gene GAPDH (Life Technologies, Carlsbad, CA, USA) and to the sham using the “-ΔΔCt method”.

Clarysse M., Accarie A., Farré R., Canovai E., Monbaliu D., Gunst J., De Hertogh G., Vanuytsel T., Pirenne J, & Ceulemans L.J. (2023). Protective Effect of Oxygen and Isoflurane in Rodent Model of Intestinal Ischemia-Reperfusion Injury. International Journal of Molecular Sciences, 24(3), 2587.

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Quantitative PCR Analysis of Gene Expression

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Total RNA was isolated from cells using the RNeasy isolation kit (Qiagen), and cDNA prepared using the Quantitech Reverse Transcription kit (Qiagen). SYBR-green-based quantitative PCR assays were deigned and optimized for HLA-C and Hprt. Samples collected from tissue such as dissected implantation sites from pregnant mice or organs were cut into small pieces and immersed in RNAlater (Qiagen) as per manufacturer’s instructions, prior to processing for RNA isolation. The sequences of the primers used for the assays are provided in Supplementary Data 10. Standard curves for each of the assays were performed using serial dilutions of cDNA and amplification efficiencies were determined. Relative expression was expressed as 2^–ΔCt, where ΔCt is the difference of the cycle threshold between the transcript of the gene of interest and the reference gene transcript.

Kaur G., Porter C.B., Ashenberg O., Lee J., Riesenfeld S.J., Hofree M., Aggelakopoulou M., Subramanian A., Kuttikkatte S.B., Attfield K.E., Desel C.A., Davies J.L., Evans H.G., Avraham-Davidi I., Nguyen L.T., Dionne D.A., Neumann A.E., Jensen L.T., Barber T.R., Soilleux E., Carrington M., McVean G., Rozenblatt-Rosen O., Regev A, & Fugger L. (2022). Mouse fetal growth restriction through parental and fetal immune gene variation and intercellular communications cascade. Nature Communications, 13, 4398.

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Quantifying Mesenchymal Markers in HUVECs

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Total RNA was isolated from HUVECs using the RNeasy Isolation kit (Qiagen, Maryland) and cDNA was generated from the mRNA template by qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD), according to the manufacturer’s instructions. Human α-SMA, N-cadherin, Snail, and GAPDH mRNA expression were quantified using the 7300 real-time PCR system and commercially available validated TaqMan^® Gene expression primers (Applied Biosystems, Foster City, CA). The relative expression of each gene was compared to GAPDH and calculated according to the 2^−ΔΔCt method, as described [16 (link)].

Woda C.B., Bruneau S., Mak A.L., Haskova Z., Liu K., Ghosh C.C, & Briscoe D.M. (2019). CALCINEURIN INHIBITORS AUGMENT ENDOTHELIAL-TO-MESENCHYMAL TRANSITION BY ENHANCING PROLIFERATION IN ASSOCIATION WITH CYTOKINE-MEDIATED ACTIVATION. Biochemical and biophysical research communications, 519(4), 667-673.

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RNA-Seq Analysis of DLBCL Tumors

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Total RNA was extracted from normal GC B cells (from human tonsils) and DLBCL tumors using Trizol (LifeTechnologies) and RNeasy isolation Kit (Qiagen). RNA concentration was determined using Qubit (LifeTechnologies) and integrity was verified using Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were generated using the TruSeq RNA sample kit (Illumina). First-strand synthesis was performed using random oligos and SuperscriptIII (Invitrogen). After second-strand synthesis, a 200-bp paired-end library was prepared following the Illumina paired-end library preparation protocol. Pair-end sequencing (PE50) was performed on Illumina HiSeq2000. RNA sequencing results were aligned to hg19, respectively, using STAR (Dobin et al., 2013 (link))and annotated to RefSeq using the Rsubread package(Liao et al., 2013 (link)).

Li M., Chiang Y.L., Lyssiotis C.A., Teater M.R., Hong J.Y., Shen H., Wang L., Hu J., Jing H., Chen Z., Jain N., Duy C., Mistry S.J., Cerchietti L., Cross J.R., Cantley L.C., Green M.R., Lin H, & Melnick A.M. (2019). Non-oncogene Addiction to SIRT3 Plays a Critical Role in Lymphomagenesis. Cancer cell, 35(6), 916-931.e9.

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Gene Expression Analysis by qRT-PCR

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mRNA was extracted from cells using the Rneasy Isolation Kit (Qiagen). According to the manufactures protocol for single-stranded cDNA synthesis, 500 ng of total RNA was reverse transcribed using iScript cDNA synthesis kit (BIO-Rad). cDNA samples were amplified using SYBR green supermix (BIO-Rad), in a white 96 multiwell plate by LightCycler 96 system instrument (Roche) according to the manufacture's protocol. To quantify the data, the comparative Ct method was used. Relative quantity was defined as 2-^ΔΔCt and β2-Microglobulin was used as reference gene. The following primers were used: CDH2 F-AGTCACCGTGGTCAAACCAATCGA, R-TGCAGTTGACTGAGGCGGGTG; SPOCK1 F-GCTCAGATGGCCACTCCTAC, R-TCTGTGCAGGCACTCCTTTC; LBH F-TCCCCATTCACTGCCCCGAC, R-AAAGGCCATCCTTGCGGGGG; FERMT1 F- CTTGGTTCAGTGACAGCCCT, R-GGAGTCTAGCCAACCTGCAT; MMP10 F-GACAGAAGATGCATCAGGCAC, R- CATCTTGCGAAAGGCGGAAC; NEDD9 F-GCTGCCGAAATGAAGTATAAGAATC, R-GGTCAGGATGTCTCCCTTGC; KDM7A F-GTCTGAATTGGTGGAGGTCCC, R- ATCTTCTCACCCCAGAGGACA; CCDC80 F-GGATCTGTGGCGGTACTCTG, R-GTGTAATCCAATGGTGGCTCA; IL11 F- ATGAACTGTGTTTGCCGCCT, R- TCAGCTGGGAATTTGTCCCTC; STX5 F- AGTCTTTGGTCGGGTTTCGG, R- CGCATAACCTCGGACTCCC. B2M F-ATGAGTATGCCTGCCGTGTG, R-GGCATCTTCAAACCTCCATG; GAPDH F-ATGGGGAAGGTGAAGGTCG, R- GGGGTCATTGATGGCAACAATA; HRPT1 F-TGACACTGGCAAAACAATGCA, R-GGTCCTTTTCACCAGCAAGCT and ACTB F- CATGTACGTTGCTATCCAGGC, R-CTCCTTAATGTCACGCACGAT.

Vervoort S.J., Lourenço A.R., Tufegdzic Vidakovic A., Mocholi E., Sandoval J.L., Rueda O.M., Frederiks C., Pals C., Peeters J.G., Caldas C., Bruna A, & Coffer P.J. (2018). SOX4 can redirect TGF-β-mediated SMAD3-transcriptional output in a context-dependent manner to promote tumorigenesis. Nucleic Acids Research, 46(18), 9578-9590.

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Quantitative Analysis of Gene Expression

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Total RNA was extracted from cells in culture using the RNeasy isolation kit (Qiagen). First-strand complementary DNA (cDNA) was synthesized from total RNA using the AMV reverse transcriptase (Roche) and a combination of random hexamers and polyA primers, according to the manufacturer’s instructions. Each of the target genes was amplified in real-time from cDNA using oligonucleotides specific to that gene (sequences and conditions available upon request), with 28S-RNA, Rpl27, and Rpl4 used as the normalization controls. cDNA levels were determined using SYBR green in a MyiQ rt-PCR detection system (BioRad). All samples were run in triplicate.

Beltrán D., Anderson M.E., Bharathy N., Settelmeyer T.P., Svalina M.N., Bajwa Z., Shern J.F., Gultekin S.H., Cuellar M.A., Yonekawa T., Keller C, & Campbell K.P. (2019). Exogenous expression of the glycosyltransferase LARGE1 restores α-dystroglycan matriglycan and laminin binding in rhabdomyosarcoma. Skeletal Muscle, 9, 11.

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Comprehensive RNA Extraction and qPCR Analysis

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Total RNA was extracted with RNeasy isolation kit (Qiagen, Valencia, CA), per manufacturer’s instructions. Dll4, Spry-2, Col I and SMA were amplified with Sso Advanced SYBR Green Supermix kit (Bio-Rad, Hercules, CA) with the primers specific for mouse genes (Table 2).Table 2

Primers used in the study

Gene	Direction	Sequence (5′–3′)
Collagen 1	Forward	TCTGACTGGAAGAGCGGAGAG
Collagen 1	Reverse	GGCACAGACGGCTGAGTAGG
Dll4	Forward	TCGAAATGGTGGCAGCTGTAAGGA
Dll4	Reverse	ATGCTCACAGTGCTGGCCATAGTA
IL10	Forward	CTCCAAGACCAAGGTGTCTAC
IL10	Reverse	GGAGTCCAGCAGACTCAATAC
IL12	Forward	GGGAGAAGCAGACCCTTACA
IL12	Reverse	TTCAGGCGGAGCTCAGATAG
PPARγ	Forward	CTGGCCTCCCTGATGAATAAAG
PPARγ	Reverse	AGGCTCCATAAAGTCACCAAAG
SPRY-2	Forward	ACTGCTCCAATGACGATGAGGACA
SPRY-2	Reverse	CCTGGCACAATTTAAGGCAACCCT
SMA	forward	TGACAGAGGCACCACTGAACC
SMA	reverse	TCCAGAGTCCAGCACAATACC AGT

Veliceasa D., Biyashev D., Qin G., Misener S., Mackie A.R., Kishore R, & Volpert O.V. (2015). Therapeutic manipulation of angiogenesis with miR-27b. Vascular Cell, 7, 6.

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Apabetalone Modulates Inflammation in HUVECs

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HUVECs were pretreated with apabetalone (5 or 20 μM) or vehicle (DMSO) for 1 h before addition of TNFα (10 ng/ml) for 4 h. HUVEC total RNA was then isolated (RNEasy isolation kit, Qiagen) and sent to NanoString for multiplex gene expression analysis (255 human genes) with the nCounter® Inflammation v2 Panel. NanoString was run by the University of Alberta pathology core and data was analyzed in house with the nSolver^TM software and Ingenuity^R Pathway Analysis software (Oct 2018 update). Genes with fold change > 1.3 and <− 1.3 were used in Ingenuity^R Pathway Analysis.

Tsujikawa L.M., Fu L., Das S., Halliday C., Rakai B.D., Stotz S.C., Sarsons C.D., Gilham D., Daze E., Wasiak S., Studer D., Rinker K.D., Sweeney M., Johansson J.O., Wong N.C, & Kulikowski E. (2019). Apabetalone (RVX-208) reduces vascular inflammation in vitro and in CVD patients by a BET-dependent epigenetic mechanism. Clinical Epigenetics, 11, 102.

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Gene Expression Analysis by qPCR

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Total RNA was isolated using RNeasy Isolation Kit according to the manufacturer's instructions (Qiagen). cDNA was synthesized with the Superscript III First-Strand Synthesis system (Invitrogen), and real-time PCR was performed on a Bio-Rad iCycler with SSOFast SYBR Green Supermix (Bio-Rad) and primer pairs specific for cDNA (5′ to 3′) were as follows: IL17A Forward: TTTAACTCCCTTGGCGCAAAA, IL17A Reverse: CTTTCCCTCCGCATTGACAC; IL17F Forward: TGCTACTGTTGATGTTGGGAC, IL17F Reverse: AATGCCCTGGTTTTGGTTGAA; CCR6 Forward: CCTGGGCAACATTATGGTGGT, CCR6 Reverse: CAGAACGGTAGGGTGAGGACA; CCL20 Forward: GCCTCTCGTACATACAGACGC, CCL20 Reverse: CCAGTTCTGCTTTGGATCAGC. Quantification of relative mRNA expression was determined by the comparative CT (critical threshold) method where the amount of target mRNA, normalized to endogenous β-actin expression, is determined by the formula 2^-ΔCT and was represented as 2^−ΔCt, where ΔCt = Ct_IL2 – Ct_Actin. In some experiments, normalized Ct values were presented as an induction relative to expression in control samples.

Sen S., He Z., Ghosh S., Dery K.J., Yang L., Zhang J, & Sun Z. (2018). Critical role of PRMT1 in Th17 differentiation by regulating the reciprocal recruitments of STAT3 and STAT5. Journal of immunology (Baltimore, Md. : 1950), 201(2), 440-450.

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