5.Activate the default RNA archive protocol to purify total RNA from the cell lysate (Figure 3).
6.If the default RNA Archive protocol is used, purified RNA will be eluted in 150 μL of PCR compatible buffer. Note: If the cell culture growth media is Ca2+/Mg2+ free, lysis may be performed directly in the growth media by addition of an equal volume of 2X lysis reagent.
Applied Biosystems total RNA lysis chemistry is specified to give the following results when used as recommended.
Yield of Total RNA (RNase-free) Total RNA recovery: > 50% for seeding densities in the range of 104 and106 cells per well (Figure 4 and 5)
This recovery rate may be routinely assessed using the Cell Lysate Control Kit, containing lysates at two concentrations: 104 and 106 cells/well. Total RNA is purified from these two lysates and compared to RNA standards included in the kit in an ABI PRISM 7700 Sequence Detection System based RT+ 18S ribosomal RNA assay.
Precision and Reproducibility of Recovery Coefficient of variation (%CV): <30%
Total experimental coefficient of variation, including 6700 workstation purification, all pipetting steps and analysis by ABI PRISM 7700 Sequence Detection System is less than 30% (Figure 4 and 6).
Purity of Total RNA
Purity: < 0.5% w/w of genomic DNA
Demonstrated performance of A260/280 ratio: >1.9 (Figure 6 and 7)
CT suppression of neat sample: ≤0.5
The presence of inhibitors after purification of total RNA samples is a widespread problem with other total RNA extraction methods. The ABI PRISM 7700 Sequence Detection System and an RT+ GAPDH based assay easily