When analyzing mRNA expression, 1 µg of RNA was retrotranscribed using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions and using an S1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA).
The absence of genomic contamination of RNA was confirmed by performing a PCR reaction on the cDNA using PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and the ATPA1 primers [25 (link)]. These primers produce a genomic-derived amplicon of 300 bp and a cDNA-derived amplicon of 180 bp, allowing for genomic DNA contamination detection.
When analyzing miRNA expression, 250 ng of RNA was retrotranscribed using miScript II RT Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions and using an S1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA).
The absence of genomic contamination of RNA was confirmed by performing a PCR reaction on the cDNA using PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and the ATPA1 primers [25 (link)]. These primers produce a genomic-derived amplicon of 300 bp and a cDNA-derived amplicon of 180 bp, allowing for genomic DNA contamination detection.
When analyzing miRNA expression, 250 ng of RNA was retrotranscribed using miScript II RT Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions and using an S1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA).
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