FlashTag™ Biotin HSR RNA Labeling Kits

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always keep thawed reagents and reaction tubes on ice

Upstream tips
Always keep thawed reagents and reaction tubes on ice

Publication protocol

For each sample, 400 ng of total RNA was labelled with the FlashTag Biotin RNA labelling kit for Affymetrix GeneChip miRNA 2.0 arrays (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. A tailing reaction was carried out at 37°C for 15 min in a volume of 15 µL reaction mix (10 µL of total RNA and spike control oligos mix, 1 × reaction buffer, 1.5 mM MnCl2, ATP, and 1 mL poly A polymerase enzyme) followed by the ligation of the biotinylated signal molecule to the target RNA sample at 25°C for 30 min (with the addition of 4 µL of 5 × FlashTag Ligation Mix Biotin and 2 µL of T4 DNA Ligase into the 23.5 mL of reaction mix). Finally, 2.5 mL of stop solution was added to stop the reaction. After labelling, the samples were hybridised on Affymetrix GeneChip miRNA 2.0 arrays (Affymetrix, Santa Clara, CA, USA) at 48°C and 60 rpm for 16 h in a volume of 103.2-µL reaction mix (which contained 21.5 µL of total biotin-labelled RNA, 50 µL hybridisation mix, 15 µL formamide, 10 µL DMSO, 5 µL eukaryotic hybridisation controls, and 1.7 µL control oligonucleotide B2). Immediately after hybridisation the arrays were washed and stained with streptavidin phycoerythrin conjugate to remove nonspecific binding. Finally, they were scanned to determine miRNA expression levels.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for FlashTag™ Biotin HSR RNA Labeling Kits below.

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