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Taqman qrt pcr assay

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TaqMan qRT-PCR assays are a type of real-time reverse transcription polymerase chain reaction (RT-PCR) assays. They are designed for the quantitative detection and analysis of specific RNA targets.

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Lab products found in correlation

Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions) Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (5 mentions) Mirneasy mini kit, by Qiagen (3 mentions) Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Taqman mirna assay, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Mirneasy kit, by Qiagen (2 mentions) Rnu48, by Thermo Fisher Scientific (2 mentions) Taqman probes and primers, by Thermo Fisher Scientific (2 mentions) Gapdh, by Thermo Fisher Scientific (2 mentions) Cel mir 39, by Qiagen (2 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Superscript 2 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions) Rneasy column, by Qiagen (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions) Stepone software, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Roche (1 mentions)

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QRT-PCR) TaqMan microRNA Assays TaqMan qRT-PCR TaqMan PCR assays QRT-PCR TaqMan assays

21 protocols using taqman qrt pcr assay

1

RNA Extraction and qRT-PCR for Inflammatory Markers

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Tissue RNA was extracted using Qiagen RNEasy Plus Mini Kit (Qiagen, CA, USA) following manufacturer’s instruction while cDNA was synthesized by Superscript™ first-strand cDNA system kit (Promega Corporation, Waltham, MA, USA). Expression of inflammatory markers were estimated by TaqMan qRT-PCR assays (Thermo Fisher Scientific, Austin, TX, USA) detailed in Supplementary Table 2.
Jose S., Mukherjee A., Abhyankar M.M., Leng L., Bucala R., Sharma D, & Madan R. (2018). Neutralization of macrophage migration inhibitory factor improves host survival after Clostridium difficile infection. Anaerobe, 53, 56-63.
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2

Gene Expression Profiling of CCND1, CCND2

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RNA concentration and integrity were assessed with an Agilent 2100 Bioanalyzer. Approximately 200 ng of total RNA were reverse-transcribed into cDNA using the SuperScript II First-Strand Synthesis kit (Thermo Fisher, California, EEUU). Gene expression of CCND1 and CCND2 were evaluated with TaqMan qRT-PCR assays, Hs00765553_m1 and Hs00153380_m1, respectively (Thermo Fisher). The PGK1 gene (Hs00943178_g1, Thermo Fisher) was used as the endogenous control. Relative expression was calculated whereby ΔCt =Ct housekeeping gene minus Ct target gene.
Cardona-Benavides I.J., Misiewicz-Krzeminska I., Rojas E.A., De Ramón C., Sanz-Solas A., Isidro I., Quwaider D., López-Guerrero A.M., Cuadrado M., Calasanz M.J., Rosiñol L., Martínez-López J., Miguel J.F., Mateos M.V., Corchete L.A, & Gutiérrez N.C. (2023). Quantification of cyclin D1 and D2 proteins in multiple myeloma identifies different expression patterns from those revealed by gene expression profiling. Haematologica, 109(3), 877-887.
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3

Quantitative miRNA and mRNA Expression Analysis

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The miRNeasy Mini Kit (Qiagen) was used for RNA extraction per manufacturer’s instructions. Quality and concentration of total RNA was assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). For mature miRNA measurements, 10–20 ng of total RNA were reverse transcribed using Taqman microRNA reverse transcription kit (Life Technologies, Foster City, CA) and the product was diluted 1:15 in RNAse-free water. Levels of mature miRNAs, as well as normalizers RNU44 and U6 snRNAs, were assayed in triplicates following quantitative real time polymerase chain reaction with Taqman qRT-PCR assays (Life Technologies) and analyzed using the formula C = 2^Ctgeomean of normalizers / 2^CtmiRNA. Both reverse transcription and qRT-PCR miRNA primers were part of Taqman miRNA assays (Life Technologies). miR-34a expression was normalized to U6 and RNU44 expression to control for the total amount of RNA present in each sample. For mRNA measurements, 300 ng of RNA were reverse transcribed using the VILO cDNA synthesis kit (Invitrogen). The geometric mean of β-actin and 18S rRNA or 18S rRNA alone was used for normalization using the following formula: C = 2^Ctnormalizers / 2^CtmRNA.
Bavamian S., Mellios N., Lalonde J., Fass D.M., Wang J., Sheridan S.D., Madison J.M., Zhou F., Rueckert E.H., Barker D., Perlis R.H., Sur M, & Haggarty S.J. (2015). Dysregulation of miR-34a Links Neuronal Development to Genetic Risk Factors for Bipolar Disorder. Molecular psychiatry, 20(5), 573-584.
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4

Dgcr8 Haploinsufficiency Modulates miRNA Levels

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All animal protocols used in this study are approved by Columbia University IACUC. Dgcr8+/− mice have been described previously (Stark, et al., 2008 (link)) and have been backcrossed into C57BL/6J background for over ten generations. After embryonic dissection, dissociated neurons from individual pups were cultured at ~2 × 106 neurons per condition. At DIV 1, cells were treated with Co(III)PPIX or DMSO (no porphyrin control) as indicated in Figure 6. Co(III)PPIX was dissolved in DMSO. At DIV 3, total RNA was isolated using miRNeasy mini kit (Qiagen). Taqman qRT-PCR assays (Life Technologies) were used to measure mature miR-185, miR-134 and Gapdh mRNA levels, as described previously (Stark, et al., 2008 (link); Xu, et al., 2013 (link)).
Barr I., Weitz S.H., Atkin T., Hsu P., Karayiorgou M., Gogos J.A., Weiss S, & Guo F. (2015). Cobalt (III) protoporphyrin activates the DGCR8 protein and can compensate microRNA processing deficiency. Chemistry & biology, 22(6), 793-802.
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5

Quantitative Real-Time PCR Analysis

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RNA was isolated using Trizol (Life Technologies) according to manufacturer’s instructions. For mRNA analysis, RNA was treated with DNAse I (Roche, Basel, Switzerland) and purified over a Qiagen (Hilden, Germany) RNeasy column. First strand synthesis was performed using a High Capacity cDNA Reverse Transcription kit (Life Technologies). qPCR was performed using SYBR green (Life Technologies) on a Step One Real Time PCR system with Step One software (Life Technologies). All primer sequences are provided in Supplementary Table 3. TBP was used for normalization. For miRNA analysis, RNA was used in Taqman qRT-PCR assays according to the manufacturer’s protocol (Life Technologies). U6 snRNA was used for normalization.
Langer E.M., Kendsersky N.D., Daniel C.J., Kuziel G.M., Pelz C., Murphy K.M., Capecchi M.R, & Sears R.C. (2017). ZEB1-repressed microRNAs inhibit autocrine signaling that promotes vascular mimicry of breast cancer cells. Oncogene, 37(8), 1005-1019.
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6

Quantitative miRNA and mRNA Expression Analysis

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The miRNeasy Mini Kit (Qiagen) was used for RNA extraction per manufacturer’s instructions. Quality and concentration of total RNA was assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). For mature miRNA measurements, 10–20 ng of total RNA were reverse transcribed using Taqman microRNA reverse transcription kit (Life Technologies, Foster City, CA) and the product was diluted 1:15 in RNAse-free water. Levels of mature miRNAs, as well as normalizers RNU44 and U6 snRNAs, were assayed in triplicates following quantitative real time polymerase chain reaction with Taqman qRT-PCR assays (Life Technologies) and analyzed using the formula C = 2^Ctgeomean of normalizers / 2^CtmiRNA. Both reverse transcription and qRT-PCR miRNA primers were part of Taqman miRNA assays (Life Technologies). miR-34a expression was normalized to U6 and RNU44 expression to control for the total amount of RNA present in each sample. For mRNA measurements, 300 ng of RNA were reverse transcribed using the VILO cDNA synthesis kit (Invitrogen). The geometric mean of β-actin and 18S rRNA or 18S rRNA alone was used for normalization using the following formula: C = 2^Ctnormalizers / 2^CtmRNA.
Bavamian S., Mellios N., Lalonde J., Fass D.M., Wang J., Sheridan S.D., Madison J.M., Zhou F., Rueckert E.H., Barker D., Perlis R.H., Sur M, & Haggarty S.J. (2015). Dysregulation of miR-34a Links Neuronal Development to Genetic Risk Factors for Bipolar Disorder. Molecular psychiatry, 20(5), 573-584.
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7

Pancreatic Cancer miRNA Expression Analysis

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At first, we examined the expressions of miR-6510 and miR-934 in two pancreatic cancer cell lines (Hs766t and Hs766t-L3) by qRT-PCR. RNA enriched for small non-coding RNAs was extracted from the cells using the miRNeasy Kit (Qiagen, Valencia, CA). The expression of miRNAs was quantified via TaqMan qRT-PCR assays (Applied Biosystems, Foster City, CA) on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Primers for qRT-PCR of miR-6510 and miR-934 were as follows in Supplementary Table 3. U6 was used as endogenous controls for data normalization. 2-ΔΔCT method was used to calculate the expression of each miRNA. Then, two GSE database GSE38781 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38781) and GSE163031 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163031) of pancreatic cancer were used to validate the influence of the expression of 4 miRNAs on the prognosis of pancreatic cancer.
Gong X., Liu Y., Zheng C., Tian P., Peng M., Pan Y, & Li X. (2022). Establishment of a 4-miRNA Prognostic Model for Risk Stratification of Patients With Pancreatic Adenocarcinoma. Frontiers in Oncology, 12, 827259.
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8

Extraction and Detection of RNA

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Extraction of total RNA from cells was in accordance to the instruction of mirVana™ miRNA Isolation Kit (Thermo Scientific). For isolation of intact RNA from tissues, tissues were minced on ice, frozen in liquid nitrogen immediately and stored at −80 °C until analyzed.
Intact RNA from tissues after homogenization was prepared using mirVana™ miRNA Isolation Kit (Thermo Scientific). Total RNAs were eluted with 100 μl of elution buffer. TaqMan microRNA assay was used to determine levels of mature miRNAs in tissues or cells according to the instruction of TaqMan microRNA reverse transcription kit (Thermofisher).
qRT-PCR is the most sensitive technique for mRNA and miRNA detection. TaqMan qRT-PCR assays were used for the detection of gene/mature miRNA expression in triplicate on an ABI 7500 system (Applied Biosystems, Foster City, CA). All the gene/miRNA-specific probes were bought from Applied Biosystems.
Wang Y., Mo Y., Wang L., Su P, & Xie Y. (2018). Let-7b contributes to hepatocellular cancer progression through Wnt/β-catenin signaling. Saudi Journal of Biological Sciences, 25(5), 953-958.
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9

Quantitative RT-PCR for AID Expression

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Quantitative RT-PCR was as described previously TaqMan qRT-PCR assays (Applied Biosystems) consisting of amplification primers and fluorescently labelled MGB-probes were used to quantify AID expression49 (link). qRTPCR was performed in triplicate for each sample with each gene multiplexed with an18s endogenous control (taqman assay labelled with different dye). Samples were run on a viia7 qPCR machine (Applied Biosystems) and subjected to ∆CT analysis relative to 18S expression.
Zhao Y., Uduman M., Siu J.H., Tull T.J., Sanderson J.D., Wu Y.C., Zhou J.Q., Petrov N., Ellis R., Todd K., Chavele K.M., Guesdon W., Vossenkamper A., Jassem W., D’Cruz D.P., Fear D.J., John S., Scheel-Toellner D., Hopkins C., Moreno E., Woodman N.L., Ciccarelli F., Heck S., Kleinstein S.H., Bemark M, & Spencer J. (2018). Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue. Nature Communications, 9, 3857.
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10

Quantitative RT-PCR for miRNA and mRNA Expression

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The procedure for qRT‐PCR has been described previously.18, 22, 29 The expression levels of miRNAs were analyzed by TaqMan qRT‐PCR assays (assay IDs 002148 and 002676; Applied Biosystems, Foster City, CA, USA). Data were normalized to the expression of RNU48 (assay ID 001006; Applied Biosystems). NCS1 expression levels were determined using TaqMan probes and primers (assay ID Hs00179522_m1; Applied Biosystems), and GAPDH (assay ID Hs99999905_m1; Applied Biosystems) was used for normalization.
Uchida A., Seki N., Mizuno K., Misono S., Yamada Y., Kikkawa N., Sanada H., Kumamoto T., Suetsugu T, & Inoue H. (2018). Involvement of dual‐strand of the miR‐144 duplex and their targets in the pathogenesis of lung squamous cell carcinoma. Cancer Science, 110(1), 420-432.
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