Low Input Quick Amp Labeling Kits

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		Label all samples at one time to avoid day-to-day variations. The labeled samples can be stored at -80°C upto 3 months and successive hybridizations

Downstream tips
Label all samples at one time to avoid day-to-day variations. The labeled samples can be stored at -80°C upto 3 months and successive hybridizations

Publication protocol

RNA extraction, amplification, and labeling
Total RNA was extracted from gill tissue using Qiagen RNeasy Kits (Qiagen, Inc., Valencia, CA, USA) according to manufacturer's instructions. RNA concentrations (ng μL−1) and purity (A260/A280 and A260/A230 ratios) were determined using a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA), and integrity was verified through electrophoresis. Two hundred nanograms of total RNA was then amplified and labeled with Cy3 fluorescent dye using the One‐Color Low Input Quick Amp Labeling kit (Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. Briefly, complementary DNA (cDNA) was made from control RNA‐spiked samples followed by complementary RNA (cRNA) synthesis, amplification and Cy3 labeling, and purification. We quantified cRNA concentration and dye incorporation using a NanoDrop ND1000 Spectrophotometer. All samples yielded at least 1.65 μg cRNA and specific activity 6 pmol Cy3 μg cRNA−1. To minimize technical artifacts, all reactions were performed simultaneously and individuals from each treatment were randomized in 96‐well plates and subsequently on microarray slides. Dye‐labeled samples were stored in amber tubes at −80°C until microarray hybridization.

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Manufacturer protocol

Download the product protocol from Agilent Technologies for Low Input Quick Amp Labeling Kits below.

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