6. First and Second Strand Synthesis (Second Round of RNA Amplification):
Use the materials and protocols provided in the primary RNA amplification kit to produce cDNA from aRNA made during the first round of RNA amplification.
7. Amino Allyl UTP Incorporation (Second Round of RNA Amplification):
Use the double-stranded cDNA as the template for a second round of amplification with simultaneous labeling using a commercial kit (Fluorescent Linear Amplification Kit, Agilent), then perform 5-(3-aminoallyl)-UTP (aaUTP) incorporation according to the manufacturers protocol.
8. Check Yield:
Quantitate 1L of aaUTP aRNA product using the NanoDrop ND-1000 Spectrophotometer according to the manufacturers specifications.
Under the Nucleic Acids module of the NanoDrop software, select RNA-40 as the constant for measuring the aaUTP aRNA. Expected yield is approximately 30-70g of total aaUTP aRNA, from a starting input of 10ng of total RNA.
Note: Due to the large amount of aRNA generated after the second round of amplification, a 2 to 4-fold dilution of final aRNA may be required for accurate reading on the ND-1000 spectrophotometer.
9. Check aaUTP Incorporation:
By measuring the amount of amino allyl that is incorporated into the aRNA, one can better estimate how well the aRNA will label with the dye. Poor incorporation of the amino allyl can result in inefficient labeling which will lead to reduced dye signal and loss of data. Assess the incorporation of aaUTP by checking the 289/260 ratio using the ND-1000. Generally, a ratio of 0.22-0.32 indicates adequate incorporation of amino allyl. In such instances that the ratio is not within this desired range, the rel