Development of a microarray platform for FFPET profiling: application to the classification of human tumors.

Author(s): Duenwald S, Zhou M, Wang Y, Lejnine S, Kulkarni A, Graves J, Smith R, Castle J, Tokiwa G, Fine B, Dai H, Fare T, Marton M

Publication: J Transl Med, 2009, Vol. 7, Page 65

PubMed ID: 19638234 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 formalin fixation and RNA quality on microarray analysis of breast and colon carcinoma specimens.

   Summary of Findings:

   In FFPE colon specimens, amplification yield was proportional to input quantity and depended on prior amplification. In the first round of reverse transcription, 100 ng FFPE breast RNA yielded 1-2 ug of cRNA, but when a second round of amplification was used, 500 ng of FFPE breast cRNA yielded 60-115 ug. The GAPDH CT correlated highly with the 3' slope log mean ratio from microarray data and the CT of APPBP1 correlated with gene expression patterns suggesting that CT maybe a good indicator of suitability of FFPE specimens for microarray analysis. Probes that were cytosine rich were particularly sensitive to RNA quality as determined by CT. When hybridizing FFPE RNA, the HumFFPET array, which contained probes targeting the 3' end, had higher sensitivity and specificity than the standard array, but both arrays were similar when RNA from frozen specimens was hybridized. Using the HumFFPET array, the correlation between matched frozen and FFPE specimens was 0.80, but when the standard array was used, the correlation was 0.70. Application of the breast cancer prognostic signature to data obtained from frozen and FFPE specimens resulted in correlations of 0.88 and 0.81, representing the good and poor prognostic categories, respectively.

   Biospecimens

   • Tissue - Breast
   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH APPB1
   DNA microarray Specific	Type of array	Standard Human 44 k v1.1 HumFFPET 44 k 2.0
   DNA microarray Specific	Template/input amount	0 ug 10 ug 50 ug 100 ug

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