GeneChip Sample Submission Requirements
To submit samples to the CFG Microarray facility for gene expression analysis, quality control information must be included with each sample. These requirements for submitting either total RNA or fragmented cRNA are described below and examples are provided in appropriate places throughout the protocols.
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To submit total RNA for cRNA synthesis and gene expression analysis, the following must be provided:
- A minimum of 12 µg total RNA resuspended in RNase-free water at a concentration ≥ 1 µg/µL.
- A list of the samples.
- An agarose gel photo (loading ~500ng of RNA for each lane) of the samples.
- OD readings (A260, A280, A260/280) for each sample.
To submit fragmented cRNA for gene expression analysis, the following must be provided:
- A minimum of 20 µg fragmented biotinylated cRNA resuspended in RNase-free water at a minimum concentration of 1 µg/µL.
- A list of the samples. 0.6 µg/µL.
- An agarose gel photo showing 1 µL of the unpurified IVT reaction and 1 µL of the purified IVT reaction and 1µl of the fragmented cRNA of each sample.
- OD readings (A260, A280, A260/280) for each of the purified IVT samples.
- Adjusted yields for the cRNA samples
For quantification of cRNA when using total RNA as starting material, an adjusted cRNA yield must be calculated to reflect carryover of unlabeled total RNA. Using an estimate of 100% carryover, use the formula below to determine adjusted cRNA yield:
adjusted cRNA yield = RNAm -(totalRNAi )(y)
RNAm = amount of cRNA measured after IVT (µg)
total RNAi = starting amount of total RNA (µg)
y = fraction of cDNA reaction used in IVT
Example: Starting with 10 µg total RNA, 50% of the cDNA reaction is added to the IVT, giving a yield of 50 µg cRNA. Therefore, adjusted cRNA yield = 50 µg cRNA - (10 µg total RNA) x(0.5 cDNA reaction) = 45.0 µg.