RNA amplification concentration

From VLP

Concentration of aRNA/cRNA
1. Add 0.5 volumes of 7.5 M NH4OAc and 2.5 volumes of absolute ethanol (stored at -20°C) to the sample and vortex.

2. Precipitate at -20°C for 1 hour to overnight.

3. Centrifuge at 12,000 x g at 4°C for 30 minutes.

4. Wash pellet twice with 0.5 mL of 80% ethanol (stored at -20°C). Air dry the pellet and check for dryness before resuspension.

5. Resuspend dried pellet in 10 to 20 µL of RNase-free water.

Note: If 1 µg cDNA template was used in the IVT reaction, expect 30 to 50 µg cRNA for one-half IVT reaction.

Step 3: Quantifying the cRNA (IVT Product)

 * Use spectrophotometric analysis to determine the cRNA yield. Apply the convention that 1 absorbance unit at 260 nm equals 40 µg/mL RNA.
 * Check the absorbance at 260 nm and 280 nm to determine sample concentration and purity.
 * Maintain the A260/A280 ratio close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable).
 * 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 - (total RNAi)(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) (0.5 cDNA reaction) = 45.0 µg.
 * Use adjusted yield in Eukaryotic Target Hybridization.