Gene specific probes

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

Gene-specific probes are laboratory tools used to detect and analyze specific DNA or RNA sequences in biological samples. They are designed to hybridize with complementary target sequences, allowing for the identification and quantification of specific genes or gene expression.

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TaqMan gene specific probes (8 citations) FAM-labeled gene-specific probes and primers (3 citations) Taqman probes for specific genes (2 citations) Universal TaqMan master mix and primer/probe sets specific for the genes (2 citations) TaqMan™ Universal PCR Master Mix with FAM-labeled gene specific probes (2 citations)

13 protocols using gene specific probes

Quantitative Gene Expression Profiling

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RNA was purified from tissue samples and cultured cells using TRIzol reagent (Invitrogen). TaqMan technology was used to determine the expression of target genes using Fast Start Essential DNA Probe Master (Roche Diagnostics, Indianapolis, IN) and gene-specific probes from Invitrogen (Carlsbad, CA). Resulting data were analyzed using the comparative cycle threshold method. TATA-binding protein (TBP) data were used as an endogenous reference to normalize gene expression levels.

Xu H., Li J., Chen H, & Ghishan F.K. (2018). NHE8 Deficiency Promotes Colitis-Associated Cancer in Mice via Expansion of Lgr5-Expressing Cells. Cellular and Molecular Gastroenterology and Hepatology, 7(1), 19-31.

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Nanostring Analysis of NTC Genes

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Dissected brain tissue from the patient and control were homogenized in TRIzol Reagent (Invitrogen). Following the manufacturer’s instructions (Invitrogen), total RNA was extracted, and gene-specific probes were designed to the coding region of all 227 NTC-related genes by the manufacturer (NanoString Technologies). In addition, probes targeting clathrin heavy chain (CLTC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucuronidase-β (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and protein kinase cGMP-dependent type I (PRKG1) served as five internal reference genes. Scientific Support Services (UCL Genomics) performed the NanoString nCounter assay using RNA samples diluted to the appropriate concentration. Raw data in reporter code count (RCC) files were loaded into nSolver and used to perform quality assessment and normalization. Fold change of NTC-related genes expression in the patient was calculated compared to the control.

Miao C., Jiang Q., Li H., Zhang Q., Bai B., Bao Y, & Zhang T. (2016). Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans. G3: Genes|Genomes|Genetics, 6(10), 3307-3316.

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MDM2-Targeting sd-rxRNAs: Dose-Response Study

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Example 2

MDM2-targeting sd-rxRNAs were tested in an in vitro dose response study. The sd-rxRNAs were tested for activity in RB177 cells (human retinoblastoma cell line) cells (50,000 cells/well, 96 well plate). RB177 cells were treated with varying concentrations of MDM2-targeting sd-rxRNAs or non-targeting control (#21803) in serum-free media. Concentrations tested were 1, 0.5, 0.1, 0.05, 0.025 and 0.01 μM. The non-targeting control sd-rxRNA (#21803) is of similar structure to the MDM2-targeting sd-rxRNA and contains similar stabilizing modifications throughout both strands. Forty eight hours post administration, cells were lysed and mRNA levels determined by the Quantigene branched DNA assay according to manufacturer's protocol using gene-specific probes (Affymetrix). FIGS. 2A-D demonstrate dose response analysis of lead MDM2 sd-rxRNAs in vitro in RB177 cells. Data were normalized to a house keeping gene (PPIB) and graphed with respect to the non-targeting control. Error bars represent the standard deviation from the mean of biological triplicates.

US11279934B2. Methods for treating cancer using nucleic acids targeting MDM2 or MYCN (2022-03-22). Phio Pharmaceuticals Corp. [US]. Inventors: Michael Byrne [US], Karen G. Bulock [US], James Cardia [US].

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MDM2-targeting sd-rxRNA Silencing Kinetics

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Example 3

The duration of action of MDM2-targeting sd-rxRNAs was tested in vitro in RB177 cells following a single administration of the sd-rxRNA. The sd-rxRNAs were tested for activity in RB177 cells (human retinoblastoma cell line—50,000 cells/well, 96 well plate) over a period of 6 days. RB177 cells were treated with varying concentrations of a panel of MDM2-targeting sd-rxRNAs or non-targeting control (#21803) in serum-free media. Concentrations tested were 1 and 0.2 μM. The non-targeting control sd-rxRNA (#21803) is of similar structure to the MDM2-targeting sd-rxRNA and contains similar stabilizing modifications throughout both strands. Media was changed every forty-eight hours. Cells were lysed on day 2, 4 or 6 post administration and mRNA levels determined by the Quantigene branched DNA assay according to manufacturer's protocol using gene-specific probes (Affymetrix). FIG. 3 demonstrates the duration of silencing of MDM2 targeting sd-rxRNAs in vitro in RB177 cells. Data were normalized to a house keeping gene (PPIB) and graphed with respect to the non-targeting control. Error bars represent the standard deviation from the mean of biological triplicates.

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Quantitative Gene and miRNA Expression

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Total RNA of tissues or cells was isolated with TRIzol reagent (Invitrogen) according to the manufacturer. RNAs were reverse transcribed into cDNA with 300 units of M-MLV reverse transcriptase (BRL, Gaithersburg, MD) after removing contaminating DNA by DNA-free DNase (Ambion, Austin, TX). For gene expression, the quantitative real-time PCR (qRT - PCR) assays were performed with gene specific probes (Applied Biosystems, Foster City, CA). GAPDH was used as an internal control.
To quantify miRNA expression, the total RNA was reverse transcribed with a miRNA-specific looped RT primer (Applied Biosystems). MiR-4728 was tested with miRNA-specific Taqman minor groove binder probes (Applied Biosystems). U6 was used as an internal control. All Taqman qRT- PCR studies were performed in triplicate on an ABI 7500 system (Applied Biosystems) and the data were presented as mean ± SE.

Liu Z., Zhang J., Gao J, & Li Y. (2018). MicroRNA-4728 mediated regulation of MAPK oncogenic signaling in papillary thyroid carcinoma. Saudi Journal of Biological Sciences, 25(5), 986-990.

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Quantifying GC-C and Uroguanylin Expression

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Gene expression was assessed using RT-PCR similar to previous reports in rat hypothalamic tissue. Briefly, RNA was extracted from tissue using trireagent, assessed for quality using a spectrophotometer, and converted to cDNA. Expression of GC-C and uroguanylin were determined using gene-specific probes in accordance with the manufacturer’s instructions (Applied Biosystems, Foster City, CA).

Begg D.P., Steinbrecher K.A., Mul J.D., Chambers A.P., Kohli R., Haller A., Cohen M.B., Woods S.C, & Seeley R.J. (2014). Effect of Guanylate Cyclase-C Activity on Energy and Glucose Homeostasis. Diabetes, 63(11), 3798-3804.

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Quantification of Gene Expression Profiles

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cDNA was synthesized using the SuperScript VILO cDNA synthesis kit (Invitrogen) or SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. The gene expression levels of Wig-1, FAS, 14-3-3σ and GAPDH were quantified on a 7500 Real-Time PCR System sequence detection system version 1.2 (Applied Biosystems, Foster City, CA, USA). Gene-specific probes (Applied Biosystems) were as follows: Wig-1 (Hs00536976_m1), 14-3-3σ (Hs00968567_s1), FAS (Hs00531110_m1) and GAPDH (Hs99999905_m1). The qRT–PCR was performed according to the manufacturer’s recommendations. Relative gene expression was calculated by the 2^–ΔΔCt method after normalization to GAPDH levels.

Bersani C., Xu L.D., Vilborg A., Lui W.O, & Wiman K.G. (2014). Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ. Oncogene, 33(35), 4407-4417.

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Quantitative Analysis of Cell Cycle Genes

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Quantitative real-time PCR was performed using the Applied Biosystems Step One Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). Gene expressions were analyzed using TaqMan^® Gene Expression Master Mix (Applied Biosystems) with 50 ng of cDNA as templates. ACTB (actin transcript) was used as an internal control. Gene-specific probes (Applied Biosystems) were as follows: CCND1 (Hs00765553_m1), CCND3 (Hs01017690_g1), CCNE2 (Hs00180319_m1), CCNA2 (Hs00996788_m1), CCNB1 (Hs01030099_m1), CCNG2 (Hs00171119_m1), CDK1 (Hs00938777_m1), CDK2 (Hs01528894_m1), CDK6 (Hs01026371_m1), ZFP36L2 (Hs00272828_m1), and ACTB (Hs01060665_g1). Relative gene expression was calculated by the relative standard curve or 2^−ΔΔdCt relative quantification methods.

Noguchi A., Adachi S., Yokota N., Hatta T., Natsume T, & Kawahara H. (2018). ZFP36L2 is a cell cycle-regulated CCCH protein necessary for DNA lesion-induced S-phase arrest. Biology Open, 7(3), bio031575.

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Quantitative gene expression analysis

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The total RNA was extracted and purified with TRIzol reagent (Life Technologies, NY, USA) according to the manufacturer’s instructions. A parallel tube without RT (RT-negative control) was included in the RT reactions and subsequent TaqMan PCR procedures as the control for possible DNA contamination.
The reverse transcription of RNA to cDNA was conducted using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). One hundred nanograms of total cDNA were added per 20 μl reaction with sequence-specific primers and Taqman probes. Quantitative gene expression was analyzed for PLA2G16 (Hs00912734_m1) and GAPDH (Hs02758991_g1) with gene-specific probes (Applied Biosystems) using Taqman Universal PCR Master Mix and was carried out in triplicate on an ABI Prism 7900 system according to the manufacturer’s instructions. The data were then quantified using the comparative Ct method for relative gene expression compared with GAPDH as endogenous control.

Liang S., Ren Z., Han X., Yang J., Shan L., Li L., Wang B., Zhang Q., Mu T., Chen K., Xiong S, & Wang G. (2015). PLA2G16 Expression in Human Osteosarcoma Is Associated with Pulmonary Metastasis and Poor Prognosis. PLoS ONE, 10(5), e0127236.

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Quantification of DYRK2 Gene Expression

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Total RNA from formalin-fixed paraffin-embedded tissues was isolated using an Allprep DNA/RNA kit (Qiagen, Tokyo, Japan). The quantity and quality of RNA were evaluated by spectrophotometry. Reverse transcription of RNA to cDNA was achieved using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative gene expression was determined for DYRK2 (Hs00705109_s1) and 18 s (Hs03928990_g1) using gene-specific probes (Applied Biosystems) using TaqMan Fast Advanced Master Mix and the 7900HT Fast Real-time PCR system (Applied Biosystems). PCR conditions were: 5 °C for 2 min and 95 °C for 20 s, followed by 45 cycles at 95 °C for 1 s and 60 °C for 20 s. Data were then quantified using the comparative Ct method for relative gene expression compared with 18S as an endogenous control.

Nomura S., Suzuki Y., Takahashi R., Terasaki M., Kimata R., Terasaki Y., Hamasaki T., Kimura G., Shimizu A, & Kondo Y. (2015). Dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) as a novel marker in T1 high-grade and T2 bladder cancer patients receiving neoadjuvant chemotherapy: DYRK2 is associated with survival in bladder cancer. BMC Urology, 15, 53.

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