Protocol detail

Validating miRNA Targeting of HLA-G

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For validation experiments, we selected the four miRNAs most likely to target the HLA-G gene (NG_029039.1) and messenger RNA (NM_002127.5) based on the sequence alignment analysis (RNAhybrid v.2.2) (Krûger, Rehmsmeier, 2006 (link)). The representative scheme showing the interaction site between HLA-G and the four miRNAs selected for validation was constructed using the ApE v2.0.61 software (https://jorgensen.biology.utah.edu/wayned/ape/). The TaqMan Advanced miRNA cDNA Synthesis Kit (Life Technologies, Foster City, California, USA), TaqMan Advanced miRNA Assay (reference: miR-191-5p; targets: miR-5096, miR-4516, miR-4488, miR-486-5p; Life Technologies), and TaqMan Fast Advanced Master Mix (Life Technologies) were used according to the manufacturer’s instructions to evaluate the miRNA expression. Reverse transcription PCR (RT-PCR) assays were performed in a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, California, USA) and quantitative PCR (q-PCR) in a QuantStudio 5 Real-Time System (Applied Biosystems) and 7500 Real-Time System (Applied Biosystems) according to the manufacturer’s instructions.

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Almeida R.S., Gomes T.T., Araújo F.S., de Oliveira S.A., Santos J.F., Donadi E.A, & Lucena-Silva N. (2022). Differentially Expressed Bone Marrow microRNAs Are Associated With Soluble HLA-G Bone Marrow Levels in Childhood Leukemia. Frontiers in Genetics, 13, 871972.

Publication 2022

TaqMan Advanced miRNA cDNA Synthesis Kit TaqMan Advanced miRNA Assay TaqMan Fast Advanced Master Mix SimpliAmp Thermal Cycler QuantStudio 5 Real-Time System 7500 Real-Time System

Assays Based sequence analysis Cdna Gene Hla g Messenger rna Mirna Reverse transcription Synthesis

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Variable analysis

independent variables

Selection of the four miRNAs most likely to target the HLA-G gene (NG_029039.1) and messenger RNA (NM_002127.5) based on the sequence alignment analysis (RNAhybrid v.2.2)

dependent variables

MiRNA expression evaluated using the TaqMan Advanced miRNA Assay (reference: miR-191-5p; targets: miR-5096, miR-4516, miR-4488, miR-486-5p)

control variables

The TaqMan Advanced miRNA cDNA Synthesis Kit (Life Technologies, Foster City, California, USA)
TaqMan Fast Advanced Master Mix (Life Technologies)
Reverse transcription PCR (RT-PCR) assays performed in a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, California, USA)
Quantitative PCR (q-PCR) performed in a QuantStudio 5 Real-Time System (Applied Biosystems) and 7500 Real-Time System (Applied Biosystems)

controls

The TaqMan Advanced miRNA Assay used miR-191-5p as a reference miRNA

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