Amino Allyl MessageAmp™ II aRNA Amplification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Some reagents may form a precipitate when stored at –20°C. If a precipitate is visible, redissolve it by warming the solution to room temperature with gentle mixing.

Upstream tips
Some reagents may form a precipitate when stored at –20°C. If a precipitate is visible, redissolve it by warming the solution to room temperature with gentle mixing.

Publication protocol

Embryo RNA isolation, amplification, and labeling
Four individual embryos from each treatment at developmental stage 31 [62] were used for RNA isolation, labeling, and microarray hybridization. Embryo RNA was extracted using a TRIzol buffer (Invitrogen, Carlsbad, CA, USA) followed by purification using the Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA).

Purified RNA was quantified with a spectrophotometer, and RNA quality was assessed by gel electrophoresis. RNA for hybridization was prepared by one round of amplification (aRNA) using Ambion’s Amino Allyl MessageAmp aRNA Kit to form copy template RNA by T7 amplification. Amino-allyl UTP was incorporated into targets during T7 transcription, and resulting amino-allyl aRNA was coupled to Cy3 and Cy5 dyes (GE Healthcare, Piscataway, NJ, USA).

Labeled aRNA samples (2 pmol dye/μl) were hybridized to slides in 10 μl of hybridization buffer [50% formamide buffer, 5x SSPE, 1% sodium dodecyl sulfate, 0.2 mg/ml bovine serum albumin, 1 mg/ml denatured salmon sperm DNA (Sigma), and 1 mg/ml RNase free poly(A) RNA (Sigma)] for 44 hours at 42°C. Slides were prepared for hybridization by blocking in 5% ethanolamine, 100 mM Tris pH 7.8, and 0.1% SDS added just before use for 30 minutes at room temperature, washed for one hour in 4x SSC, 0.1% SDS at 50°C, and then boiled for 2 minutes in distilled water to denature the cDNAs. Resulting 16 bit Tiff Images were quantified using ImaGene® (Biodiscovery, Inc.) spotfinding software. Controls and any gene that did not have at least one individual with a signal greater than the average signal from all herring sperm control spots (non-specific hybridization signal) plus one standard deviation were removed prior to statistical analysis. In total, 6,754 genes were analyzed.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Amino Allyl MessageAmp™ II aRNA Amplification Kit below.

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