cDNA synthesis

1. Use 15 |g total RNA for first and second strand cDNA synthesis according to manufacturer's protocol.

2. Clean up the cDNA using Phase Lock Gel tubes according to manufacturer's protocol.

3. Generate labeled cDNA by IVT using the ENZO BioArray kit according to manufacturer's protocol.

4. Clean up the cRNA with Qiagen RNeasy columns according to manufacturer's protocol.

5. Quantify the cRNA: measure Abs260nm of cRNA (typically diluted 1/50 to 1/100) to determine the yield. When using total RNA as the starting material, it is necessary to calculate an adjusted cRNA yield to correct the carryover of unlabeled total RNA. Using an estimate of 100% carryover, use the following formula to determine the adjusted cRNA yield:

adjusted cRNA yield = RNAm - (total RNA)

RNAm = amount of cRNA measured after IVT (|g)

RNA; = starting amount of total RNA (|g)

Note: use the adjusted cRNA yield when calculating the amount of cRNA needed for fragmentation and array hybridization.

6. Check 1 |g of the purified transcripts on a 1% agarose gel. Transcript lengths should range from 0.5 to 2 kb.

7. Fragmentation of the cRNA. Mix the following: 16 |g cRNA (Note: use adjusted cRNA yield.)

8 |l 5 x fragmentation buffer RNAse-free H2O to 40 |l

Incubate at 95°C for 35 min. Store at -20°C. Check 2.5 |l (=1 |g) on a 1% agarose gel. The size of the cRNA should be reduced to 100 bp.

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