Optimized protocol for gene expression analysis in formalin-fixed, paraffin-embedded tissue using real-time quantitative polymerase chain reaction.

Author(s): Votavova H, Forsterova K, Stritesky J, Velenska Z, Trneny M

Publication: Diagn Mol Pathol, 2009, Vol. 18, Page 176-82

PubMed ID: 19704263 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 RNA extraction method, hydration solution, and real-time RT-PCR labeling method on RT-PCR success using FFPE large B-cell lymphomas and to compare amplification to that using RNA extracted from fresh leukocytes. The guanidine/phenol/chloroform (GPC) RNA extraction method included deparaffinization in xylol, ethanol rehydration, digestion in proteinase K, and incubation in denaturation solution at 55 degrees C overnight, followed by phenol-chloroform extraction and rehydration in RNase-free water or Tris EDTA (TE).

   Summary of Findings:

   RNA extracted using the High Pure kit reached the exponential phase of B2M and HPRT amplification at least 5 cycles before RNA extracted using GPC. Hydration solution did not impact the B2M CT values for RNA extracted using GPC. Obtaining an RNA yield of more than 5600 ng predicted PCR success, but RNA concentration, absorbance at 260 or 280 nM, or the ratio of the absorbance at 260/280 nM were not predictive PCR success. Only 2 of the 8 specimens isolated with the High Pure kit had successful amplification of B2M and HPRT RNA without primer dimers when SYBR green was use for detection. B2M was successfully amplified in all 8 High Pure-extracted specimens, and HPRT was successfully amplified in 4 of 8 High Pure-extracted specimens when probes were used for detection. The authors report that predeveloped probe sets had a slightly higher amplification success rate than house-designed probe sets. Using the High Pure kit for RNA extraction, RevertAid for reverse transcription and TaqMan probes for detection of B2M and ribosomal protein P0 (RPLP0) led to successful amplification in 62 of 65 specimens with the three failures from specimens with less than 5600 ng of RNA extracted. While the average B2M and HPRT CTs were much lower using fresh specimens than FFPE specimens (7-8 cycles), the delta CTs were similar.

   Biospecimens

   • Tissue - Lymph Node
   • Cell - White Cells

   Preservative Types

   • Formalin
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Lymphoma
   • Not specified

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh)
   Analyte Extraction and Purification	Analyte isolation method	High Pure RNA Paraffin kit GPC method
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	Water TE TE and heating at 70 degrees C for 10 min
   Biospecimen Acquisition	Biospecimen location	Large B-cell lymphoma Leukocytes
   Real-time qRT-PCR Specific	Targeted nucleic acid	B2M HPRT1 RPLP0
   Real-time qRT-PCR Specific	Detection method	House-designed TaqMan probes Predeveloped TaqMan probes SYBR green

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2. Study Purpose

   The purpose o f this study was to determine the effects of reverse transcriptase on amplification of B2M, HPRT and TBP RNA from fresh leukocytes.

   Summary of Findings:

   RNA that was reverse transcribed with Transcriptor allowed for successful amplification of B2M, HPRT and TBP in each of 4 specimens, and RNA that was reverse transcribed with RevertAid allowed for successful amplification of B2M and TBP in all 4 specimens and HPRT in 3 of 4 specimens. In contrast, while RNA from all 5 specimens that was reverse transcribed by M-MLV had successful amplification of B2M, 3 and 4 of 5 samples had successful amplification of HPRT and TBP, respectively.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   RT-PCR Specific	Targeted nucleic acid	B2M HPRT1 TBP
   RT-PCR Specific	Reaction solution	Transcriptor RevertAid M-MLV

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