If starting from FFPE material it is recommended to use Cofactor Genomics' ImmunoPrism FFPE RNA Extraction Kit, since this will help to maximize RNA quality. RNA degradation is often an issue with FFPE samples and any steps taken to improve RNA quality/integrity will be useful for ensuring a greater likelihood of a useable RNA sample. We recommend starting with two slides of 10 micron thickness for FFPE samples with a cross-sectional area between 50 and 100 mm^2 and 4 slides of 10 micron thickness for samples with an area between 10 and 50 mm^2.
For isolating RNA from fresh, frozen tissue there are generally fewer issues with obtaining intact (high RIN) RNA, though we do recommend you start with at least 10mg of tissue.
For intact RNA you will need at least 20ng of total RNA and at least 40 ng of RNA for more degraded samples (RIN < 6.0). No matter how much RNA is available, you will need RNA samples with DV200 > 20%.
There are separate ImmunoPrism library preparation procedures for highly degraded RNA (RIN 1 or 2) - which uses Steps 1B and 2B (this will NOT include fragmentation), and partially degraded (RIN 2-7) or intact RNA (RIN > 7), either of which will use Steps 1A and 2A (this will include fragmentation).
If you are having any difficult with obtaining samples that meet these thresholds, feel free to reach out to: