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Taqman rt pcr microrna assays

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TaqMan RT-PCR microRNA assays are a set of laboratory instruments designed for the detection and quantification of microRNA expression levels. These assays utilize real-time reverse transcription-polymerase chain reaction (RT-PCR) technology to accurately measure the abundance of specific microRNA molecules in a given sample.

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Lab products found in correlation

Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (3 mentions) Mirna taqman probe, by Thermo Fisher Scientific (1 mentions) Oligo d t 16 primer, by Thermo Fisher Scientific (1 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)

3 protocols using taqman rt pcr microrna assays

1

Quantifying mRNA and miRNA Levels in Human Brain Tissue

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Total RNA (including miRNA fraction) was isolated from human postmortem frozen brain tissue as we reported previously (4 (link)). Total mRNA was reverse transcribed using M-MLV reverse transcriptase and oligo (dT) 16 primers. miRNA was reverse transcribed using TaqMan RT-PCR microRNA assays (Table S2; Applied Biosystems, Toronto, ON, Canada). Real-time PCR reactions were run in technical quadruplets using the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Toronto, ON, Canada). Expression levels were calculated using the Absolute Quantitation (AQ) standard curve method, with GAPDH used as the reference gene for mRNA quantification and RNU6B for miRNA. The efficiencies of RT-PCR ranged between 90% and 110%, with slopes between −3.10 to −3.50. See details in Supplemental Materials and Methods.
Torres-Berrío A., Lopez J.P., Bagot R.C., Nouel D., Dal-Bo G., Cuesta S., Zhu L., Manitt C., Eng C., Cooper H., Storch F., Turecki G., Nestler E.J, & Flores C. (2016). DCC confers susceptibility to depression-like behaviors in humans and mice and is regulated by miR-218. Biological psychiatry, 81(4), 306-315.
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2

Validating Small RNA Sequencing with qRT-PCR

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Small RNA sequencing data was validated using qRT-PCR. Total RNA samples were reverse transcribed using TaqMan RT-PCR microRNA assays (Applied Biosystems) according to the manufacturer’s instructions. Real-time PCR reactions were run in quadruplicate using the ABI 7900HT Fast Real-Time PCR System and data was collected using the Sequence Detection System 2.4 (SDS) software (Applied Biosystems). Expression of miRNAs was quantified using miRNA TaqMan probes (Applied Biosystems) and calculated using the Absolute Quantitation (AQ) standard curve method. RNU6B was used as an endogenous control as it showed expression levels that remained relatively constant with low variance and high abundance across the samples tested.
Lopez J.P., Diallo A., Cruceanu C., Fiori L.M., Laboissiere S., Guillet I., Fontaine J., Ragoussis J., Benes V., Turecki G, & Ernst C. (2015). Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing. BMC Medical Genomics, 8, 35.
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3

Quantification of mRNA and miRNA Expression

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Total mRNA was reverse–transcribed using M–MLV reverse transcriptase (Gibco) and oligo(dT)16 primers (Invitrogen). miRNA was reverse transcribed using TaqMan RT–PCR microRNA assays (Applied Biosystems) according to the manufacturer’s instructions. Real–time PCR reactions were run in quadruplets using the ABI 7900HT Fast Real–Time PCR System and data was collected using the Sequence Detection System (SDS) software (Applied Biosystems). To measure miRNA expression, we used miRNA TaqMan probes, considered to be the gold standard for miRNA quantification33 (link). Expression levels were calculated using the Absolute Quantitation (AQ) standard curve method, with β–Actin and GAPDH used as endogenous controls for mRNA quantification. Five endogenous controls were tested for miRNA quantification: U6, RNU6B, RNU44, U47 and RNU48 but only RNU6B was selected as it showed expression levels that remained relatively constant with low variance and high abundance across samples tested. All primers used in the study can be found in Supplementary Table. 8.
Lopez J.P., Lim R., Cruceanu C., Crapper L., Fasano C., Labonte B., Maussion G., Yang J.P., Yerko V., Vigneault E., Mestikawy S.E., Mechawar N., Pavlidis P, & Turecki G. (2014). miR-1202: A Primate Specific and Brain Enriched miRNA Involved in Major Depression and Antidepressant Treatment. Nature medicine, 20(7), 764-768.
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