Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.
Author(s): Patel PG, Selvarajah S, Boursalie S, How NE, Ejdelman J, Guerard KP, Bartlett JM, Lapointe J, Park PC, Okello JB, Berman DM
Publication: J Vis Exp, 2016, Vol. , Page 54299
PubMed ID: 27583817 PubMed Review Paper? No
Purpose of Paper
This paper investigated how nucleic acid yields are affected by Proteinase K digestion (source and duration) and storage of formalin-fixed paraffin-embedded (FFPE) tissue cores. The suitability of FFPE tumors for methylation studies and the impact of using autopsy specimens on RNA yield and expression were also investigated.
Conclusion of Paper
The authors developed an extraction protocol that was successfully applied to FFPE benign liver tumor specimens, and brain, bladder and breast cancer specimens. Digestion with high potency Proteinase K resulted in increased DNA yield, while extending the duration of Proteinase K digestion from 2 h to 24 h resulted in nonsignificant increases in DNA yield. Storage duration of FFPE cores did not affect DNA or RNA yields. Bisulfite converted DNA isolated from FFPE specimens displayed little variation in ALU methylation and the expected increase in GSTP1 methylation in prostate cancer specimens but not benign specimens. RNA yields were higher from surgical specimens than autopsy specimens, and while RT-PCR analysis was successful for surgical tissue and postmortem liver specimens, amplification was not successful for postmortem brain specimens.
Studies
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Study Purpose
This study investigated how nucleic acid yields are affected by the type and duration of Proteinase K digestion, and storage of FFPE tissue cores. The suitability of FFPE tumors for methylation studies and the impact of using autopsy specimens on RNA yield and expression were also investigated. Details on specimens processing and handling were not specified. Effects of the duration of Proteinase K digestion on DNA and RNA yields were investigated using three FFPE specimens of unspecified origin. Effects of FFPE storage were investigated using 333 prostate FFPE cores that were collected between 2000 and 2012. Differences between tissue types and autopsy and surgical specimens were investigated using one benign liver specimen and eight brain tumor specimens obtained at autopsy and two bladder and three breast tumor surgical resection specimens, respectively. DNA and RNA were extracted from 3 FFPE cores using the AllPrep DNA/RNA FFPE kit and an optimized protocol that included deparaffinization in xylene, homogenization in ethanol, digestion with Proteinase K at 56˚C for 15 min with agitation, and centrifugation to isolate RNA from DNA. The RNA containing supernatant was incubated at 80˚C for 15 min and purified with a spin column. DNA was extracted by additional Proteinase K digestion at 56˚C for 24 h, a 2 h incubation at 90˚C and spin column purification. DNA and RNA were quantified fluorometrically.
Summary of Findings:
Digestion with a high potency Proteinase K, as opposed to the temperature stable Proteinase K supplied in the AllPrep DNA/RNA FFPE kit, increased DNA yield. Extending Proteinase K digestion from 2 h to 24 h resulted in non-significant increases in DNA yield, but the authors report more consistent yields with digestion for 24 h. The duration of specimen storage was not significantly correlated with DNA or RNA yield, but DNA and RNA yields were significantly and weakly correlated to each other (r=0.39, P<0.0001). More than twice as much RNA was recovered as DNA from FFPE specimens. The optimized extraction protocol was successfully applied to FFPE benign liver tumors, and brain, bladder and breast cancers. The authors state that RNA yields were higher from surgical than autopsy specimens and while RT-PCR analysis was successful for surgical tissues and the postmortem liver specimens, amplification was not successful for postmortem brain specimens. Bisulfite converted DNA from FFPE specimens displayed little variation in ALU methylation and the expected increase in GSTP1 methylation in prostate cancer specimens but not benign specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Not specified
- Autopsy
- Neoplastic - Benign
Platform:
Analyte Technology Platform DNA Fluorometry DNA PCR RNA Real-time qRT-PCR RNA Fluorometry DNA Bisulfite conversion assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 3-14 years
Preaquisition Diagnosis/ patient condition Benign
Cancer
Preaquisition Postmortem interval Unspecified
0 h
Analyte Extraction and Purification Protein digestion 2 h
24 h
48 h
Temperature-stable (AllPrep) Proteinase K
High potency Proteinase K