18s rna

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18S RNA is a component of the small subunit of eukaryotic ribosomes. It plays a crucial role in the translation of mRNA into proteins within cells. This ribosomal RNA is commonly used as a genetic marker in various scientific applications, such as phylogenetic analysis and molecular biology research.

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17 protocols using 18s rna

Real-time PCR mRNA Expression Analysis

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For mRNA expression analysis, real-time PCR was performed with TaqMan® Gene Expression Assays (Applied Biosystems, AB). Real-time PCR was run on a 7500 Fast Real-Time PCR System) using TaqMan® Fast Universal PCR Master Mix (Applied BiosystemsTM) under standard conditions. Transcript levels were quantified using 18 S RNA (Applied Biosystems) as a reference and normalized.

Steiner S., Seleznik G.M., Reding T., Stopic M., Lenggenhager D., ten Buren E., Eshmuminov D., Endhardt K., Hagedorn C., Heidenblut A.M., Bratus-Neuenschwander A., Grossmann J., Trachsel C., Jabbar K.S., Hahn S.A., Berg J.V., Graf R, & Gupta A. (2022). De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer. Oncogene, 41(10), 1507-1517.

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One-step RT-qPCR for Mouse Gene Expression

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Primers and probes for mouse AREG, αSMA, procollagen I, TERT, α6 integrin and 18s RNA were purchased from Applied Biosystems. One-step real-time RT-PCR (qRT-PCR) was performed on a GeneAmp 7500 Sequence Detection System (Applied Biosystems). Results were expressed as 2^−ΔΔC_T as previously described using 18s RNA as the reference, and the respective control group as calibrator (33 (link)).

Ding L., Liu T., Wu Z., Hu B., Nakashima T., Ullenbruch M., De Los Santos F.G, & Phan S.H. (2016). Bone Marrow CD11c+ Cell-Derived Amphiregulin Promotes Pulmonary Fibrosis. Journal of immunology (Baltimore, Md. : 1950), 197(1), 303-312.

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Quantifying M1 and M2 gene expression

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THP-1 cells were cultured in the presence of media for M0, IFNγ (20 ng/mL, Peprotech) and LPS (100 ng/mL) for M1, or IL-4 and IL-13 (both at 20 ng/mL, Peprotech) for M2 or with SSI QBECO drug product (1 : 20 or 1 : 500). After 18 hrs, RNA was extracted from cell pellets of polarized THP-1 cells using a PureLink RNA Mini Kit according to manufacturer's instructions (Ambion, Life Technologies). Genomic DNA was removed and cDNA was synthesized from 100 ng RNA using QuantiTect Reverse Transcription Kit (Qiagen). Real time PCR was performed with Taqman Fast Advanced Master Mix (Applied Biosystems) in the presence of 6-carboxyfluorescein- (FAM-) labeled primers for M1 genes (CCL19, CXCL11, CCR7, and TNF) and M2 genes (CCL13, CCL18, and FGL) (Applied Biosystems) using a StepOnePlus instrument (Applied Biosystems). Amplification conditions were 95°C for 20 seconds (s), 95° for 1 s before 60°C for 20 s for 40 cycles. Expression of M1 and M2 genes was normalized to 18SRNA (Applied Biosystems) for quantification. The housekeeping gene CT value was subtracted from the gene of interest CT (ΔCT). The difference was then calculated between the control (M2) and the other samples (ΔΔCT). The fold change was then calculated from this number (2^(−ΔΔCT)).

Bressler B., Bethel K.P., Kleef R., Reynolds S.L., Sutcliffe S., Mullins D.W, & Gunn H. (2015). Site-Specific Immunomodulator: A Novel Treatment for Crohn's Disease. Gastroenterology Research and Practice, 2015, 231243.

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Quantifying Pik3ip1 mRNA Levels in Uterine Tissue

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The RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA) was
utilized in order to extract RNA from uterine tissues. The mRNA levels of
Pik3ip1 were measured through real-time PCR TaqMan
analysis, utilizing the Applied Biosystems StepOnePlus system (Applied
Biosystems, Foster City, CA, USA). Pre-validated proves, primers, 18S RNA, and
Universal Master mix reagent were purchased from Applied Biosystems (Applied
Biosystems, Carlsbad, CA). The template cDNA was made with MMLV Reverse
Transcriptase (Invitrogen Corp., Carlsbad, CA) and 1 μg of total RNA
with use of random hexamers. The real-time PCR was all done with three
independent RNA sets, and mRNA quantities were normalized against the 18S RNA
with use of the ABI rRNA control reagents. Statistical analyses were performed
using Student’s t-tests using the Instat package from GraphPad (San
Diego, CA). p<0.05 was considered statistically significant.

Teasley H.E., Chang H.J., Kim T.H., Ku B.J, & Jeong J.W. (2017). Expression of PIK3IP1 in the murine uterus during early pregnancy. Biochemical and biophysical research communications, 495(4), 2553-2558.

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Quantitative PCR Analysis of Adipose Genes

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The expression of mRNA was examined by quantitative PCR analysis using a Quantstudio PCR system (Life Technologies). Taqman assays were used to quantitate Adipoq (Mm00456425_m1), Adrb3 (Mm02601819_g1), Cd68 (Mm03047340_m1), Dio2 (Mm00515664_m1), Emr1 (F4/80) (Mm00802530_m1), Fabp4 (Mm00445880_m1), Itgam (Cd11b) (Mm00434455_m1), Leptin (Mm00434759_m1), Mapk8 (Mm00489514_m1), Mapk9 (Mm00444231_m1), Nova1 (Mm01289097_m1), Nova2 (Mm01324153_m1), Ppargc1a (Mm00447183_m1), Ppargc1b (Mm00504720_m1), Prdm16 (Mm00712556_m1), Ucp1 (Mm01244861_m1) mRNA and 18S RNA (4308329) (Applied Biosystems). Standard curves were constructed using the threshold cycle (Ct) values for each template dilution plotted as a function of the logarithm of the amount of input template. The number of mRNA copies for each gene-sample combination was calculated using the slope of the standard curve. To obtain a normalized abundance, copy numbers were corrected for the amount of 18S RNA in each sample.

Vernia S., Edwards Y.J., Han M.S., Cavanagh-Kyros J., Barrett T., Kim J.K, & Davis R.J. (2016). An alternative splicing program promotes adipose tissue thermogenesis. eLife, 5, e17672.

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Quantification of Skin Collagen and Gene Expression

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The collagen content of the skin was determined by Sircol Collagen Assay kit (Biocolor, Newtown Abbey, UK).[21 (link)] Total protein assay (Bio-Rad Laboratories, Hercules, CA) was used as control to normalize collagen content of each sample.
Tissue mRNA levels were determined by real-time quantitative PCR (RT-PCR). Total RNA was isolated from skin frozen in RNA Later (Qiagen Sciences, Maryland, MA) and purified with RNA mini kit (Qiagen Sciences, Maryland, MA). Real-time PCR was performed using validated TaqMan Gene Expression Assays for Cdh11, Col1α1, alpha-smooth muscle actin (αSMA), CCN2 (formerly called connective tissue growth factor), fibronectin, TGF-β1, IL-6, and 18s RNA (Applied Biosystems). The 18s RNA gene was used as a control to normalize transcript levels of mRNA in each sample. Data are presented as fold change.

Pedroza M., Welschhans R.L, & Agarwal S.K. (2017). Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model. PLoS ONE, 12(11), e0187109.

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Basal Cell Cytokine Expression in HIV

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To assess the effect of HIV on basal cell cytokine gene expression, basal cells (5 × 10⁴) were exposed to increasing HIV input (p24 from 5 to 200 ng/ml in 300 μl) for 2 days. Heat-inactivated HIV was used as a control. Total RNA was extracted using Trizol reagent (Invitrogen) and the aqueous phase was purified using an RNAEasy MinElute RNA purification kit (Qiagen). RNA concentration was determined using a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies). First-strand cDNA was synthesized from 0.5 µg of total RNA using TaqMan Reverse Transcription Reagents with random hexamer as primer (Applied Biosystems). All samples were analyzed in triplicate at cDNA dilution of 1:10. All reactions were run on an Applied Biosystems Sequence Detection System 7500 and relative expression levels determined using the dCt method with 18S ribosomal RNA as an endogenous control. The primers were obtained from Applied Biosystems, including IL-8 (Hs00174103_m1), GM-CSF (Hs00929873_m1), ICAM-1 (Hs00164932_m1), IL-1β (Hs00174097_m1) and 18s RNA (Hs99999901_s1). All primers were optimized for TaqMan PCR and referenced in recent studies^{56 (link)–60}.

Chung N.P., Khan K.M., Kaner R.J., O’Beirne S.L, & Crystal R.G. (2021). HIV induces airway basal progenitor cells to adopt an inflammatory phenotype. Scientific Reports, 11, 3988.

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Quantitative RT-PCR of Gene Expression

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Total RNA was extracted from cells with Trizol (Life Technology). cDNA libraries were prepared using SuperScript First-Strand Synthesis System for RT-PCR (Life Technology).
Validated Taqman assays were purchased for quantification of GAPDH (Applied Biosystems Hs 02758991), c-Myc (Applied Biosystems Hs00153408), and 18sRNA
(Applied Biosystems Hs 99999901). qRT-PCR were run and analyzed in Stepone Plus PCR system (Applied Biosystems).

Xie J., Wei X., & Chen Y. (2021). Loss of PABPC1 is compensated by elevated PABPC4 and correlates with transcriptome changes.

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Apoptosis Pathway Gene Expression

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Briefly, the cells were plated into 6-well culture dishes (3 × 10⁵ cells/well) for 24 h prior to the addition of MtRV plant extract (1.5 mg mL⁻¹). The total RNA isolation kit (A&A Biotechnology, Gdynia, Poland) was used to isolate total RNA from cells treated with the plant extracts. The obtained RNA was transcribed into cDNA using TranScriba Kit (A&A Biotechnology). Following this, the expression of four genes (Bax, Bcl-2, Cas-3, TP53) was measured by qRT-PCR using TaqMan^® Real-Time PCR Master Mix (Life Technologies, Carlsbad, CA, USA) and Agilent Technologies Stratagene Mx300SP (Santa Clara, CA, USA) working on MxPro software. TaqMan probes (Life Technologies, CA, USA) were used to analyse genes and 18S RNA (Life Technologies) was included as a reference gene. The PCR was performed as follows: 95 °C for 10 min, 30 cycles of 95 °C for 15 s and 60 °C for 60 s.

Kowalczyk T., Sitarek P., Skała E., Toma M., Wielanek M., Pytel D., Wieczfińska J., Szemraj J, & Śliwiński T. (2019). Induction of apoptosis by in vitro and in vivo plant extracts derived from Menyanthes trifoliata L. in human cancer cells. Cytotechnology, 71(1), 165-180.

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Analyzing RNA and Telomerase in Lung Fibrosis

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Total RNA was isolated using Trizol (Invitrogen). The Taqman primers for mouse procollagen I, α-SMA, TERT and 18s RNA were purchased from Life Technologies. 18s RNA was used as reference to normalize the input RNA. One-step real-time RT-PCR was performed on a GeneAmp 7500 Sequence Detection System (Applied Biosystems). Results were expressed as 2^-ΔΔCT using the indicated control group as calibrator.
Telomerase activity in the lung tissue or isolated MLF was assayed using a telomerase PCR ELISA kit (Roche) in accordance with the manufacturer’s protocol. The heat-inactivated tissue or cell lysates were used for the negative controls.

Liu T., Yu H., Ding L., Wu Z., Gonzalez De Los Santos F., Liu J., Ullenbruch M., Hu B., Martins V, & Phan S.H. (2015). Conditional Knockout of Telomerase Reverse Transcriptase in Mesenchymal Cells Impairs Mouse Pulmonary Fibrosis. PLoS ONE, 10(11), e0142547.

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