A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues.

Author(s): Takano EA, Mikeska T, Dobrovic A, Byrne DJ, Fox SB

Publication: BMC Biotechnol, 2010, Vol. 10, Page 89

PubMed ID: 21162754 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 extraction method, storage duration, and PCR fragment size and platform on the amplification of TATA box binding protein (TBP) RNA from FFPE breast carcinoma specimens. Tumor cells were microdissected from 1-5 freshly cut, deparaffinized, methyl green-stained sections, and 3 replicate extractions were performed with each method.

   Summary of Findings:

   The average RNA yield was slightly higher when RNA was extracted from FFPE tissue using the Trizol method (2.76 ug) compared to the High Pure kit (2.23 ug). The average absorbance 260/280 ratios were 1.7 and 1.8, respectively, for RNA extracted with the Trizol method and the High Pure kit. While the 300 bp fragment of TBP was not amplifiable in any of the specimens, amplification of the 92, 161 and 252 bp products was possible in at least one specimen 3, 8 and 13 years old, but amplification success rates decreased with increasing fragment size. The highest rates of successful amplification for all three products were in specimens 8 years old rather than 3 or 13 years old. More specimens extracted using the High Pure kit had CT values below 35 than specimens extracted using the Trizol method (78 and 72 of 90, respectively). Lack of successful amplification of the 92 bp fragment in the multiplex RT-PCR assay was associated with real-time PCR CT values above 35, but in 135 of 139 specimens from which there was successful amplification of the 92 bp fragment, a CT of less than 35 was obtained by real-time PCR. Further, the average real-time PCR CT decreased as the maximum amplicon size obtained in multiplex PCR increased.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	RT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	High Pure RNA Lysis, proteinase k digestion, Trizol extraction and DNAse treatment
   RT-PCR Specific	Length of gene fragment	92 bp 161 bp 252 bp
   RT-PCR Specific	Targeted nucleic acid	TBP
   Real-time qRT-PCR Specific	Targeted nucleic acid	TBP
   Storage	Storage duration	3 years 8 years 13 years
   RT-PCR Specific	Technology platform	Real-time PCR

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