The final yield depends on several variables such as the age of the sample, the quality of the sample, the fixation process, storage conditions, etc. Final yield variation can be observed between samples.
Several factors can influence the yield recovery - specifically:
RNA content: Depending on the sample type and sample age, RNA content might be lower than expected.
Sample preparation: Fixation process (duration, temperature) and delayed embedding can lead to sample degradation resulting to lower yields. Large tissue fragments can also lead to inhomogeneous tissue preservation and result in lower yields.
Sample storage: FFPE samples should be kept at 2-8°C or -20 to -70°C for longer storage period.
Input amount used for the RNA extraction.
Suboptimal preparation of the column.
Presence of paraffin in the sample: We recommend to remove as much as possible surrounding paraffin in the sample before starting the extraction. If paraffin is still visible in the aqueous phase, heat again the sample to melt the paraffin and centrifuge.
Centrifugation speeds.
Sample loading on the column: it is important to slowly load the sample in the center of the matrix to not disturb the matrix. Do not touch the matrix with the pipet tip. This can damage the matrix and generate short-circuit current which can result in introduction of lysis components into the sample and reduce yields.