Whole genome and transcriptome amplification: practicable tools for sustainable tissue biobanking?
Author(s): von Teichman A, Storz M, Dettwiler S, Moch H, Schraml P
Publication: Virchows Arch, 2012, Vol. 461, Page 571-80
PubMed ID: 23007645 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of using formalin-fixed and paraffin-embedded (FFPE) specimens rather than frozen ones for whole genome amplification (WGA), whole transcriptome amplification (WTA) and mutational analysis in specimens with known Von Hippel-Lindau (VHL) mutations.
Conclusion of Paper
GenomePlex amplification of DNA and RNA from frozen specimens resulted in 300- to 1,291-fold and 202- to 423-fold enrichments, respectively, but WGA of DNA from FFPE tissue only resulted in a 10-to 73-fold enrichment. Amplification of the 271 bp fragment of VHL was only possible using RNA from 2 of the 4 FFPE specimens, and the 350 bp fragment was not amplified using RNA from any of the 4 FFPE specimens, so the authors did not attempt WTA with RNA from FFPE specimens. Only DNA from 70% and 67% of frozen and FFPE specimens, respectively, allowed for correct mutation identification after a single round of WGA, but 3 of the FFPE specimens were assigned as having a different mutation, alone or in combination with the actual mutation, while false mutations were not identified in frozen specimens. A second round of WGA allowed for correct assignment of all frozen specimens and all but 1 of the FFPE specimens. Only 13 of the 20 frozen specimens allowed for confirmation of the mutation after a single round of WTA, but after a second round of WTA, 5 were properly assigned, but two that did not have analyzable sequencing after the first round were improperly classified as having no mutation.
Studies
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Study Purpose
The purpose of this study was to determine the effects of using FFPE specimens rather than frozen ones for WGA and mutational analysis in renal cell carcinoma specimens with known VHL mutations. The specimens were stored 9-19 years before DNA extraction. DNA was extracted from FFPE and frozen sections using the DNeasy kits, and WGA was performed using the GenomePlex kits.
Summary of Findings:
GenomePlex amplification of DNA from frozen specimens produced fragments that were 100-1,000 bp in length and resulted in a 300- to 1,291-fold enrichment, but only a 10- to 73-fold enrichment of DNA from FFPE specimens was observed, and there was more variability in yield between FFPE specimens than between frozen specimens. Only DNA from 14 of the 20 (70%) frozen specimens allowed for correct mutation identification after a single round of WGA, but after an additional round of WGA, all mutations were correctly assigned. Similarly, DNA from 8 of the 12 (67%) FFPE specimens allowed for accurate mutation identification after a single round of WGA, but the 4 discrepant cases consisted of 2 specimens showing the correct mutation and an additional mutation, one showing just a different mutation, and one had no mutation identified. After an additional round of WGA on the 4 discrepant cases, 3 were correctly assigned and the fourth went from being classified as having the correct mutation and an additional mutation to being classified as not having any mutations.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform DNA Whole genome amplification DNA Spectrophotometry DNA DNA sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Whole genome amplification Specific Nucleic acid amplification 1 round
2 rounds
-
Study Purpose
The purpose of this study was to determine the effects of using FFPE specimens rather than frozen ones for WTA and mutational analysis in renal cell carcinoma specimens with known VHL mutations. The specimens were stored 9-19 years before RNA extraction. RNA was extracted from FFPE and frozen sections using the RNeasy kits, and WTA was performed using the GenomePlex kit.
Summary of Findings:
Amplification of the 271 bp fragment of VHL was only possible using RNA from 2 of the 4 FFPE specimens, and the 350 bp fragment was not amplified using RNA from any of the 4 FFPE specimens. GenomePlex amplification of RNA from frozen specimens produced fragments of 100-1,000 bp in length and resulted in a 202- to 423-fold enrichment, but due to poor amplification of the 350 bp fragment of the VHL gene, WTA was not attempted using RNA from FFPE tissues. Only 13 of the 20 frozen specimens allowed for confirmation of the mutation after a single amplification. Of the remaining 7 specimens, 3 had sequences that were unable to be analyzed, 2 did not amplify, and 2 were improperly assigned as having no mutation. After a second round of WTA on the 7 specimens producing discrepant results, 5 were properly assigned, but two that were unable to be analyzed by sequencing after the first round were improperly classified as having no mutation.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA DNA sequencing RNA RT-PCR RNA Whole transcriptome amplification Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
RT-PCR Specific Length of gene fragment 271 bp
350 bp
Whole transcriptome amplification Specific Nucleic acid amplification 1 round
2 rounds