Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens.

Author(s): Hamatani K, Eguchi H, Takahashi K, Koyama K, Mukai M, Ito R, Taga M, Yasui W, Nakachi K

Publication: J Histochem Cytochem, 2006, Vol. 54, Page 773-80

PubMed ID: 16517976 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of rehydration temperature, duration and solution on the amplification of BCR and N-ras from FFPE thyroid cancer specimens.

   Summary of Findings:

   When RNA was preheated, higher temperature and longer duration resulted in decreased high molecular weight RNA yield. The highest yield was found when RNA was preheated at 70 degrees C for 45 min. The pH of the rehydration solution only had an effect for the longest fragments tested (98 bp for N-ras and 127 bp for BCR), and amplification was best when the buffer had a pH of 4. High efficiency amplification of N-ras and BCR was observed when a 10 mM citrate solution was used, but amplification was poor when a 50 mM solution was used.

   Biospecimens

   • Tissue - Thyroid Gland

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Incubation duration/condition	10 min 15 min 20 min 30 min 45 min 60 min 90 min 120 min
   RT-PCR Specific	Length of gene fragment	61 bp 91 bp 98 bp 121 bp 127 bp 148 bp 152 bp 199 bp 221 bp 222 bp 250 bp 275 bp
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	2 mM citrate buffer 10 mM citrate buffer pH 3-6.5 50 mM citrate buffer TE pH 7.0 60 degrees C 65 degrees C 70 degrees C 75 degrees C 80 degrees C 85 degrees C 90 degrees C 95 degrees C

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