Rna isolation kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, United Kingdom

The RNA isolation kit is a laboratory tool designed to extract and purify ribonucleic acid (RNA) from biological samples. It provides a standardized and efficient method to isolate RNA for further analysis and applications.

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Lab products found in correlation

Close spelling variants of this product

PicoPure RNA Isolation Kit (454 citations) TRIzol Max Bacterial RNA Isolation Kit (57 citations) PARIS™ Kit Protein and RNA Isolation System (34 citations) MagMax Total RNA isolation kit (28 citations) PureLink FFPE RNA Isolation Kit (27 citations) Micro Total RNA Isolation Kit (5 citations) Protein and RNA isolation system kit (5 citations) MagMAX-96 RNA Isolation kit (5 citations) RNA isolation and cDNA synthesis kits (3 citations) MoBio PowerMicrobiome RNA Isolation kit (3 citations)

161 protocols using rna isolation kit

RNA Extraction and qRT-PCR Analysis

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All samples were frozen in liquid nitrogen and stored at −80 °C for RNA extraction and other analyses. Total RNA was extracted from flower buds, leaves, roots, and stems using the RNA Isolation Kit (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol, and stored at −80 °C. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
First–strand cDNA was synthesized from 1 μg of total RNA using a Prime Script RT Reagent Kit (Takara, Dalian, China). PCR reactions were performed using the ABI 7500 Fast Real–Time PCR system (Applied Biosystems, Foster City, California, USA) and the QuantiFast SYBR Green PCR Kit (Qiagen, Duesseldorf, Germany). The amplification parameters were 95 °C for 5 min, followed by 45 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The mRNA expression levels were normalized to the level of SmActin expression using the 2^−ΔΔCt method [49 (link)]. Each experiment included three biological replicates. The primer sequences are listed in Table S1.

Chen J., Wang S., Wu F., Wei M., Li J, & Yang F. (2022). Genome–Wide Identification and Functional Characterization of Auxin Response Factor (ARF) Genes in Eggplant. International Journal of Molecular Sciences, 23(11), 6219.

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RNA Extraction and Purity Assessment

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RNA extraction was achieved using an RNA Isolation kit (Ambion, Inc, Austin, TX. USA). Gel electrophoresis followed by ethidium bromide staining was utilized to assess the RNA integrity, and the ratio between RNA absorbance at 260 nm and its absorbance at 280 nm was calculated to evaluate RNA purity.

Yang L., Yang B., Wang Y., Liu T., He Z., Zhao H., Xie L, & Mu H. (2019). The CTIP‐mediated repair of TNF‐α‐induced DNA double‐strand break was impaired by miR‐130b in cervical cancer cell. Cell Biochemistry and Function, 37(7), 534-544.

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eIF3d Knockdown Transcriptome Analysis

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RNA sequencing technology was provided by Shanghai OE Biotech Co. Ltd. Jurkat cells that had been treated with eIF3d si-RNA or control si-RNA for 48 h were used for RNA extraction using an RNA Isolation Kit (Ambion) following the manufacturer’s protocol. Libraries were constructed using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina) according to the manufacturer’s instructions. Then, these libraries were sequenced on the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X Ten), generating 125-bp/150-bp paired-end reads. Raw data (raw reads) were processed using the Next-Generation Sequencing (NGS) Quality Control (QC) Toolkit. Clean reads were mapped to a reference genome using hisat2. The Fragments Per kb Per Million Reads (FPKM) value for each gene was calculated using cufflinks, and the read counts for each gene were obtained by htseq-count. IPA pathway enrichment analysis was applied for functional categorization of differences in gene expression (absolute fold change > 1.2 and P value < 0.05).

Pan Y., Zhang Z.N., Yin L.B., Fu Y.J., Jiang Y.J, & Shang H. (2019). Reduced eIF3d accelerates HIV disease progression by attenuating CD8+ T cell function. Journal of Translational Medicine, 17, 167.

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Quantitative RT-qPCR Analysis of MTMR3, p27 mRNA

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Total RNAs were extracted using an RNA isolation kit (Ambion). The reverse transcription reaction was conducted using PrimeScript™ RT Reagent Kit (Takara, Japan) and qPCR was conducted with 2 × TaqMan Premix Ex Taq (Takara, Japan). β-actin was included for normalization. The 2^−(ΔΔCt) method was used to calculate the relative fold change of mRNA expression8 . The genes quantified were MTMR3 (primers, 5′-AGTGTCAAGAGTGGCTGAAGAG-3′ and 5′-ATAGACCTCCATGCA CCAAGC-3′; probe, 5′-FAM-TGAACAACGCAATCCGACCACCT–Eclipse-3′), p27 (primers, 5′-TGTGTAGAAAGTGAAATCAGAGGAG-3′ and 5′-CAGGAGTGATAT TATCTGGGTAAGC-3′; probe, 5′-FAM–CGGACCTCG GACAGGTGATCCACC-Eclipse-3′) and β-actin (primers, 5′-GACTACCTCATGAA GATCCTCACC-3′ and 5′-TCTCCTTAATGTCACGCACGATT-3′; probe 5′-FAM-CGGCTACAGCTTCACCA CCACGGC-TAMRA-3′).

Gong Y., He T., Yang L., Yang G., Chen Y, & Zhang X. (2015). The role of miR-100 in regulating apoptosis of breast cancer cells. Scientific Reports, 5, 11650.

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Quantifying miR-1 Transcriptional Targets

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At 36 h after miR-1 transfection of cancer stem cells, total RNAs were extracted from the cells using an RNA isolation kit (Ambion, USA). Subsequently, the first-strand cDNA was synthesized by reverse transcription with a PrimeScript first-strand cDNA synthesis kit (Takara Bio, Japan). Quantitative real-time PCR was conducted with gene-specific primers (GAPDH [glyceraldehyde-3-phosphate dehydrogenase], 5′-GGACCTGACCTGCCGTCTAG-3′ and 5′-GTAGC CCAGGATGCCCTTGA-3′; COX1, 5′-GAGAAA TGAATGAGCCTACAGA-3′ and 5′-TACACCCTAGACCAAACTTACG-3′; ND1, 5′-CGATTCCGCTACGACCAACT-3′ and 5′-AG GTTTGAGGGGGAATGCTG-3′; ATP6, 5′-GGGCGCA GTGATTATAGGCT-3′ and 5′-TAAGGGGTGTAGGTGTGCCT-3′; GPD2, 5′-GGCTTCCAGATACCCTTCCTT-3′ and 5′-TGTT GATGTTCAGCGTGTATTAGAG-3′; MINOS1, 5′-AGGATTCTTCCCCTGCTAATA-3′ and 5′-GAACTGCTT CCACCTCGTAAT-3′; ND4, 5′-ACAAGCTCCATCTGC CTACG-3′ and 5′-GCTTCAGGGGGTTTGGATGA-3′). The reaction mixture contained 5 μL of SYBR Green PCR Master Mix (Takara Bio, Japan), 0.4 μL of 10 μM forward and reverse primers, and 1 μL of cDNA at a final volume of 10 μL. The PCR was carried out at 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. GAPDH was used as an internal standard for normalization.

Zhang S., Liu C, & Zhang X. (2019). Mitochondrial Damage Mediated by miR-1 Overexpression in Cancer Stem Cells. Molecular Therapy. Nucleic Acids, 18, 938-953.

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Quantifying mRNA Expression with Real-Time PCR

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Total RNAs were extracted from cells using an RNA Isolation Kit (Ambion, USA). The reverse transcription reaction was conducted with PrimeScript RT Reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using 2 × ChamQ SYBR qPCR Master Mix (Vazyme, USA). The PCR reaction mixture (10 μL) contained Rox reference Dye, cDNA, ChamQ SYBR qPCR Master Mix (Vazyme), and primers (Table S1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene for normalization. The 2^-(△△Ct) method was used to calculate the relative fold change of mRNA expression [20 (link)]. PCR was conducted by maintaining the reaction at 95 °C for 30 s and then alternating for 40 cycles between 95 °C for 5 s and 60 °C for 30 s.

Chu M., Wan H, & Zhang X. (2021). Requirement of splicing factor hnRNP A2B1 for tumorigenesis of melanoma stem cells. Stem Cell Research & Therapy, 12, 90.

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Quantification of Tissue and Urinary miR-21

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The RNA isolation kit was purchased from Ambion, Inc. The methods of RNA extraction have been described previously.¹⁹ (link) Briefly, ten 10-μm sections were cut from the fresh frozen tissue blocks of the residual sample from the kidney biopsy using a microtome, pooled in a 1.5-mL microcentrifuge tube, and homogenized and dissolved in digestion buffer.²⁰ (link) Urine samples were centrifuged at 3,000g for 30 minutes and 13,000g for 5 minutes at 4 °C. Specimens were then stored at −80 °C until use.
Tissue and urinary miR-21 levels were quantified using real-time quantitative polymerase chain reaction (RT-QPCR) using the ABI Prism 7900 Sequence Detection System (Applied Biosystems). Commercially available Taqman primers and probes, including 2 unlabeled PCR primers and 1 FAM dye-labeled TaqMan MGB probe, were used (all from Applied Biosystems). RNU48 (Applied Biosystems) was used as housekeeping genes.²¹ (link)^,²² (link) All RT-QPCR experiments were performed in triplicate. Results were analyzed using Sequence Detection Software, version 2.0 (Applied Biosystems). The ΔΔCT method for relative quantitation was used. Urinary miR-21 level represents the total amount excreted in an 8-hour overnight urine specimen, as compared with that of the housekeeping gene.

Szeto C.C., Ng J.K., Fung W.W., Luk C.C., Wang G., Chow K.M., Lai K.B., Li P.K, & Lai F.M. (2020). Kidney microRNA-21 Expression and Kidney Function in IgA Nephropathy. Kidney Medicine, 3(1), 76-82.e1.

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Gene Expression Analysis by RT-qPCR

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Total RNA was extracted using an RNA isolation kit (Ambion). The reverse transcription reaction was conducted using a PrimeScript™ RT Reagent Kit (Takara, Japan). Quantitative real-time PCR was performed with 2× TaqMan Premix Ex Taq (Takara, Japan) to detect genes. Real-time PCR was performed according to our previous report 24 (link). The sequences of the primer pairs are shown in Supplementary Table 1.

Yang F., Chen S., He S., Huo Q., Hu Y, & Xie N. (2020). YB-1 interplays with ERα to regulate the stemness and differentiation of ER-positive breast cancer stem cells. Theranostics, 10(8), 3816-3832.

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Overexpression of TTK in HeLa Cells

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Total RNA was extracted from HeLa cells using an RNA Isolation Kit (Ambion, Austin, TX, USA). The reverse transcription reaction was conducted using PrimeScript RT Reagent Kit (Takara, Kyoto, Japan), and the cDNA was then used as the template for the amplification of TTK gene. The ORF of TTK was amplified by the following sequences: pcDNA‐Flag‐TTK‐F:GGAggatccATGGATTACAAGGACG ACGATGACAAGATGGAATCCGAGGATTTAAGTG.PcDNA‐Flag‐TTK‐R:GGTctcgagTCAAAAAGTCTTGGAGGATGAA. The fragment was then digested and cloned into the plasmid (Invitrogen, Carlsbad, CA, USA). GES‐1 was used for the transfection of Flag‐TTK.

Huang H., Yang Y., Zhang W., Liu X, & Yang G. (2020). TTK regulates proliferation and apoptosis of gastric cancer cells through the Akt‐mTOR pathway. FEBS Open Bio, 10(8), 1542-1549.

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RNA Isolation and RT-PCR Analysis

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RNA was extracted using the RNA isolation Kit (Ambion, Foster City, CA, USA), total RNA (1 μg) was reverse transcribed to cDNA by using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The primer sequences used for the reactions are in Supplementary Figure S7. RT-PCR was performed in LC480 (Roche) Sequence Detection System. GAPDH was used as the reference gene.

Lee E., Yang J., Ku M., Kim N.H., Park Y., Park C.B., Suh J.S., Park E.S., Yook J.I., Mills G.B., Huh Y.M, & Cheong J.H. (2015). Metabolic stress induces a Wnt-dependent cancer stem cell-like state transition. Cell Death & Disease, 6(7), e1805-.

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