Total RNA was purified by miRNAeasy (Qiagen, Germantown, MD, USA) and reverse-transcribed using TaqMan UNIVERSAL MMixII (Applied Biosystems, Waltham, MA, USA) for random priming or MicroRNA (miRNA)-specific assay reverse transcription. Semiquantitative PCR was performed with TaqMan-validated assays (Applied Biosystems): miR34a (000426), hsa-miR-34b (000427), miR34c (000428), hsa-miR-34c-3p (241009_mat). As reference for cDNA, we chose GAPDH (Hs99999905_m1) and U6 (#001973) for miRNA. All analyses were carried out in triplicate. Real-time data were collected using Microsoft Excel, and analyzed with the following formula: Expression level = 2−ΔΔCt method. All experiments were done as independent triplicates and analyzed using standard deviation (SD). The p-value was obtained with the Student’s t-test.
Taqman validated assays
Manufactured by Thermo Fisher Scientific
TaqMan-validated assays are a type of molecular biology tool used for the detection and quantification of specific DNA or RNA sequences. These assays utilize TaqMan probe technology, which combines the principle of fluorescence resonance energy transfer (FRET) with the 5' nuclease activity of Taq DNA polymerase. The assays provide a standardized and validated approach for sensitive and reliable target detection, making them a useful tool for various applications in genomic research and molecular diagnostics.
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3 protocols using taqman validated assays
1
Quantitative Real-Time PCR of miR-34 Family
2
RNA Extraction and qPCR Analysis
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Total RNA was purified by miRNAeasy (Qiagen, Germantown, MD, USA) and reverse‐transcribed using TaqMan UNIVERSAL MMix II (Applied Biosystems, Waltham, MA, USA) for random priming or miRNA‐specific assay reverse transcription. Semi‐quantitative PCR was performed with TaqMan‐validated assays (Applied Biosystems). As endogenous reference gene for cDNA, we chose U6 (#001973) for miRNA. All analyses were carried out in triplicate. Real‐time data were collected using Microsoft Excel and analysed with the following formula: Expression level = 2‐ΔΔCt method.17 (link) All experiments were done as independent triplicates and analysed using standard deviation (SD). The p value was obtained with Student's t test.
3
Quantifying miR-34 Family Expression
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Total RNA was purified by miRNeasy (Qiagen, Germantown, MD, USA) and reverse-transcribed by TaqMan Universal Mix II (Applied Biosystems, Waltham, MA, USA) using miRNA-specific assay reverse transcription. Semiquantitative PCR was performed with TaqMan-validated assays (Applied Biosystems): miR34a (000426), hsa-miR34b (000427), miR34c (000428), and hsa-miR34c-3p (241009_mat). As reference for cDNA, we chose U6 (#001973) for miRNA. All analyses were carried out in triplicate. Real-time data were collected using Microsoft Excel and analyzed with the following formula: expression level = 2-ΔΔCt method. All experiments were done as independent triplicates and analyzed using standard deviation (SD). The p-value was obtained with the Student’s t-test.
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