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)].
Micro rna transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Micro-RNA transcription kit is a laboratory product designed to facilitate the in vitro transcription of micro-RNA molecules. It provides the necessary reagents and protocols to generate micro-RNA from DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using micro rna transcription kit
1
Validation of miRNA Expression Profiling
2
Quantifying miR-221, miR-222, and miR-1303 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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 .
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