Labeling Protocol for Illumina Gene Expression Array

-Using Illumina TotalPrep RNA Amplification Kit

I.                  Reverse Transcription to Synthesize First Strand cDNA

1. Bring RNA samples to 11 ul with nuclease-free water.
2. Make Reverse Transcription Master Mix according to the Ambion Master Mix Calculator for Illumina TotalPrep Kit.  www.ambion.com/techlib/append/mm_calcs/illumina_rna_totalprep
3. Add 9 ul of Reverse Transcription Master Mix to each sample, vortex, and centrifuge briefly. 
4. Incubate samples at 42⁰C for 2 hours using a PCR Thermocycler.

II.               Second Strand cDNA Synthesis

1. Prepare Second Strand Master Mix on ice according to the same calculator.
2. Add 80 ul of Second Strand Master Mix to each sample, vortex, and centrifuge briefly.
3. Incubate at 16⁰C for 2 hours, with the PCR Thermocycler lid open, the samples are covered by a tip box lid.

III.           cDNA Purification

1. Preheat nuclease-free water to 52.5 ⁰C for at least 10 min.
2. Prewarm the cDNA binding buffer at 37⁰C for 10 min., cool to room temp. before use.
3. Add 24 ml ETOH to the Wash Buffer bottle.
4. Prepare and label all the tubes necessary.
5. Add 250 ul of cDNA Binding Buffer to each sample.
6. Pass the mixture through a cDNA Filter Cartridge.
7. Wash with 500 ul Wash Buffer.
8. Elute cDNA with 10 ul and 9.5 ul of 52.5⁰C Nuclease-free water respectively.

IV.           In Vitro Transcription to Synthesize cRNA

1. Make IVT Master Mix according to the same calculator.
2. Add 7.5 ul of IVT Master Mix to each cDNA sample and mix.
3. Incubate at 37⁰C for 14 hours.
4. Add 75 ul of Nuclease-free water to each sample to stop the reaction, mix and centrifuge briefly.

V.               cRNA Purification

1. Preheat Nuclease-free water to 58⁰C.
2. Assemble cRNA filter cartridge and tubes.
3. Add 350 ul cRNA Binding buffer to each sample.
4. Add 250 ul 100% ethanol and pipet 6 times to mix.
5. Pass samples through a cRNA filter cartridge.
6. Wash with 650 ul Wash buffer.
7. Elute cRNA with 90 ul preheated nuclease-free water.
8. Check the concentration and quality of the cRNA by Nano Drop.