4. First Round RNA Amplification:
Estimated total RNA quantity is used to determine the appropriate kit for RNA amplification (see Table 1).
5. Check Yield (optional):
Note: Checking the yield after one round of amplification is optional and serves the purpose of assessing the success of the first round of amplification only. It is recommended to use all of the material generated after the first round of amplification to process through a second round of amplification in order to generate microgram quantities of aRNA. Yield and quality assessment after the first round of amplification may significantly reduce the end product yield when starting with very limited material.
Quantitate 1 L of amplified RNA (aRNA) product using the NanoDrop ND-1000 Spectrophotometer according to the manufacturers specifications.
5.1 Using the Nucleic Acids module of the NanoDrop software, select RNA-40 as the constant for measuring aRNA.
5.2 For optimal results, thoroughly mix and briefly spin down the aRNA sample prior to removing 1 L from the top of the solution. This step will prevent interference caused by potential residual silica from the column purification procedure.
5.3 The yield can be theoretically calculated based on starting cell input. Example of yield assessment: With a starting cell input of 1000 cells and assuming ~10pg of RNA per cell, one can expect a yield of ~10ng of total RNA. Message content in 10ng of total RNA will be app