Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT).

Author(s): Loudig O, Milova E, Brandwein-Gensler M, Massimi A, Belbin TJ, Childs G, Singer RH, Rohan T, Prystowsky MB

Publication: Nucleic Acids Res, 2007, Vol. 35, Page e94

PubMed ID: 17636051 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare FFPE and frozen specimens and determine the effects of repair of FFPE RNA by CT-RT on the resulting fragment size, RT-PCR amplification rates, and microarray analysis. Paired frozen and FFPE breast carcinomas that had been stored for 10 years and 3 year old cervical carcinomas were compared.

   Summary of Findings:

   During the CT-RT reaction, FFPE RNA was repaired by annealing a T7 primer sequence to FFPE cDNA and hybridizing it to a reference library for reverse transcription followed by in vitro transcription with T7 polymerase. RNA from 10 year old FFPE specimens was highly degraded with most fragments less than 200 nucleotides (nt) long. In contrast, after CT-RT cRNA was 50-850 nt long, but the RNA was still much more fragmented than the 1000-2000 nt fragments obtained from 10 year old frozen specimens. Frozen specimens allowed for amplification of all three fragments of p53, cyclin D1 (CCND1), and human epidermal growth factor receptor 2 (HER-2), but a large number of non-specific products were also obtained. From non-restored FFPE RNA, no fragments larger than 150 bp were amplified for any of the three genes, but from CT-RT restored FFPE RNA, all products were amplified specifically and confirmed by sequencing. A high degree of correlation was found between technical replicates when RNA from frozen specimens was hybridized to microarrays, but the correlation was much lower when non-restored FFPE RNA was hybridized. After restoration, the correlation between technical replicates from FFPE specimens increased, and the number of good spots increased from 1306 to 4583, which compared favorably with the 4535 spots in frozen specimens. Restoration increased the correlation of microarray analysis results between RNA from FFPE and frozen specimens and led to 3 times more overlap in transcripts detected. Still, using restored RNA from FFPE specimens led to the identification of a lot fewer breast-specific and cervical-specific transcripts compared to RNA from frozen specimens.

   Biospecimens

   • Tissue - Breast
   • Tissue - Cervix

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	DNA sequencing
   RNA	RT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen Formalin (buffered)
   Biospecimen Acquisition	Biospecimen location	Breast Cervix
   RT-PCR Specific	Targeted nucleic acid	HER-2 p53 CCND1
   RT-PCR Specific	Length of gene fragment	~100 bp ~150 bp ~300 bp
   RT-PCR Specific	Template modification	Complementary-template reverse-transcription repaired RNA Non-repaired RNA

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