In the stage 2 (Validation study), the expression of the selected miRNAs (hsa-miR-155-5p, MIMAT0000646; hsa-miR-181a, MIMAT0000256; and hsa-let-7a-5p, MIMAT 0000062) was measured by stem loop TaqMan RT-qPCR (Life Technologies, Carlsbad, CA, USA). Specific stem loop primers were used to synthetize the cDNA from 10 ng total RNA using the micro-RNA transcription kit (Life Technologies, CA, USA). RT-qPCR assays were carried out using predesigned assays (miR-155, ID: 002623; miR-181a, ID: 00048; and let-7a, ID: 000377) (Life Technologies CA, USA). RNU24 was selected as the most stable endogenous reference among 4 small nucleolar RNAs (RNU24, RNU6B, RNU58, RNU44), which were evaluated by using the GeNorm software [19 (link)]. All samples were assayed in duplicate, and the relative miRNA expression was analyzed using the comparative CT method using the formula 2−ΔCt [20 (link)].
Taqman rt qpcr
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan RT-qPCR is a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) system. It is designed for the detection and quantification of RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Micro rna transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Lightcycler480 real time qpcr machine, by Roche (1 mentions)
Snorna202, by Thermo Fisher Scientific (1 mentions)
Qiazol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 mastermix, by Roche (1 mentions)
5 protocols using taqman rt qpcr
1
Validation of miRNA Expression Profiling
2
Quantifying miR-221, miR-222, and miR-1303 Expression
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miR-221 and miR-222 (MI00000298 and MI00000299, respectively) were selected from
previous reports from the literature whereas miR-1303 (MI0006370) was chosen using
bioinformatics tools. Predictive analysis using TargetScan (www.targetscan.org ) showed that miR-1303 is a conserved miR with a
high score for mRNA-miR interaction (context score percentile: 99) and this
prediction was further confirmed using the miRNA target prediction databases miRDB
(www.mirdb.org ) and Target Miner (www.isical.ac.in/~bioinfo_miu ).
miR relative quantification was performed by stem loop TaqMan® RT-qPCR (Life
Technologies, CA, USA). Specific stem loop primers were used to synthetize the cDNA
from 10 ng of total RNA using the micro-RNA transcription kit (Life Technologies, CA,
USA). Predesigned assays were purchased from Life Technologies to perform the
relative quantification using the RNU24 as an endogenous reference. RNU24 was
selected as the most stable endogenous reference among 4 small nucleolar RNAs (RNU24,
RNU6B, RNU58, RNU44), which were evaluated by using the geNorm software10 .
previous reports from the literature whereas miR-1303 (MI0006370) was chosen using
bioinformatics tools. Predictive analysis using TargetScan (
high score for mRNA-miR interaction (context score percentile: 99) and this
prediction was further confirmed using the miRNA target prediction databases miRDB
(
miR relative quantification was performed by stem loop TaqMan® RT-qPCR (Life
Technologies, CA, USA). Specific stem loop primers were used to synthetize the cDNA
from 10 ng of total RNA using the micro-RNA transcription kit (Life Technologies, CA,
USA). Predesigned assays were purchased from Life Technologies to perform the
relative quantification using the RNU24 as an endogenous reference. RNU24 was
selected as the most stable endogenous reference among 4 small nucleolar RNAs (RNU24,
RNU6B, RNU58, RNU44), which were evaluated by using the geNorm software10 .
3
Quantification of miR-224 and miR-452 by RT-qPCR
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PCR was performed as previously described (Goto et al, 2014a (link); Goto et al, 2015a (link); Kurozumi et al, 2016 (link)). The expression levels of miR-224 (Assay ID: 002099) and miR-452 (Assay ID: 002329) were analysed by TaqMan RT-qPCR (TaqMan MicroRNA Assay; Applied Biosystems) and normalised to RNU48 (Assay ID: 001006). TaqMan probes and primers for WWP1 (P/N: Hs00366931_g1) and GUSB (P/N: Hs00939627_m1) as an internal control were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).
4
Quantification of miRNA Expression
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Selected miRNAs were quantified with TaqMan RT‐qPCR according to the manufacturer's protocol (Applied BioSystems). Each reaction was primed using a gene‐specific stem‐loop primer (Applied BioSystems). The qPCR reactions were carried out by LightCycler® 480 Real‐Time PCR System (Roche Applied Science, Penzberg, Germany). Raw data were analyzed with the automatic quantification cycle setting for assigning baseline and quantification for quantification cycle determination. Relative expression of the mature miRNAs was normalized to the internal control small nuclear U6 expression (in tissue) or cel‐miRNA‐39 (in plasma) and calculated by the 2−ΔΔCT method.17 , 18 All experiments were performed in triplicate.
5
Quantification of miRNA and mRNA
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