RNA from Archived FFPE Blocks – A Valuable Underexploited Resource
Theresa J Kokkat, Virginia A LiVolsi, Miral Patel, Diane McGarvey, Munia Z Islam, Gina Piermatteo, Zubair W Baloch. University of Pennsylvania, Cooperative Human Tissue Network (Eastern Division), Philadelphia, PA
Background: Formalin Fixed and Paraffin Embedded (FFPE) tissue blocks constitute a valuable underexploited resource for molecular studies of disease. However the major limitation of FFPE samples for their use in molecular analyses is the degradation of RNA in FFPE samples. RNA Degradation can be caused 1) by various sample handling procedures prior to formalin fixation or 2) during formalin fixation which causes significant chemical modification of RNA or 3) after dehydration and paraffin-embedding where RNA continues to fragment and degrade over time.The quality of RNA can be represented either as RNA Integrity Number (RIN; Agilent Bioanalyzer) or RNA Quality Indicator (RQI; Biorad Experion) with a range of 1 to 10, representing totally degraded RNA to completely intact RNA. In FFPE samples, because RNA continues to degrade over time into small fragments, the RIN or RQI is much lower than frozen tissues. Recent improvements in the field of nucleic acid extraction as well as gene expression profiling have made it possible to study long-term archived tissues. Even a RIN of 1.4 has been successfully used for gene expression analysis. Therefore we investigated if time (in terms of years) of storage has effect on the quality of RNA on FFPE blocks. We compared RNA quality from FFPE blocks stored for 10 years, 5 years, 1 year to the blocks that have been stored for less than a year.
Design: Lung FFPE blocks (10 years, 5 years, 1 year and less than a year, n=16) were obtained from either clinically archived or the Cooperative Human Tissue Network repository. Two to four 10 um curls were cut from each block and processed using Allprep DNA/RNA FFPE Kit from Qiagen. RNA quality was investigated using automated electrophoresis. A minimum RQI of 1.4 was considered as threshold for useful RNA quality.
|Years Stored||# blocks analyzed||# cases >1.4 RQI||Mean ± S.D||Range|
|10 years||5||4||2.30 ± 1.10||1.0 -3.5|
|5 years||4||3||2.43 ± 1.55||1.0- 4.6|
|1 year||3||3||1.80 ± 0.00||Not Applicable|
|less than 1 year||4||4||2.58 ± 0.61||1.9 -2.8|