Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue.

Author(s): Green TM, de Stricker K, Møller MB

Publication: Diagn Mol Pathol, 2009, Vol. 18, Page 243-9

PubMed ID: 19861891 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to evaluate RNA quality obtained from frozen versus formalin fixed paraffin embedded lymph nodes archived 5-11 years.

   Summary of Findings:

   From 20 mg of paraffin embedded lymph nodes the authors were able to obtain between 14-446.7 ng/ul RNA with a 260/280 absorbance of 1.85-2.01. From 8 mg frozen lymph nodes 19-332.8 ng/ul RNA with a 260/280 absorbance of 2.03-2.11. The authors conclude that both methods allow for isolation of RNA of good purity and sufficient quantity.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Lymphoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen Formalin (buffered)

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2. Study Purpose

   The purpose of this study was to determine if FFPE tissues differ in terms of real-time RT- PCR amplified reference genes compared to case-matched frozen tissue and the effect of amplicon size.

   Summary of Findings:

   The ability to amplify transcripts by real-time RT-PCR varied between fixation methods and with amplicon length. Cycle threshold (Ct) values obtained from FFPE samples were 3-10 Ct values higher then those from frozen tissue indicating preservative dependent variance in the amplification of different transcripts. The authors speculate this is due to differences in degradation sensitivity between transcripts. It was found that in matched frozen and FFPE specimens transcript normalization with beta-glucoronidase (GUSB) and TATA box binding protein (TBP) together resulted in a pearson coefficient of 0.93 (P<0.01). Adding a third normalizer gene resulted in a decreased pearson coefficient. Pearson coefficients of 0.74 and 0.83 were obtained for comparisons between amplicons of different sizes of the same target transcript for frozen and FFPE samples, respectively. It was noted that the change in ranking of normalizer amplicons between preservative methods and the differential effect of size means any real-time PCR assay used in a study with multiple specimen preservation methods must be validated for each preservation method and may require the use of multiple normalizer genes. Preservation method also differentially affects amplification of amplicons of differing length so multiple target amplicons may also be necessary.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Lymphoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	ABL GUSB B2M PRKG1 GAPDH TBP
   Real-time qRT-PCR Specific	Length of gene fragment	53 bp 62 bp 63 bp 68 bp 69 bp 72 bp 76 bp 81 bp 82 bp 88 bp 92 bp 105 bp

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