PMID- 24412525
OWN - NLM
STAT- MEDLINE
DCOM- 20141028
LR  - 20140217
IS  - 1943-7811 (Electronic)
IS  - 1525-1578 (Linking)
VI  - 16
IP  - 2
DP  - 2014 Mar
TI  - Separate quality-control measures are necessary for estimation of RNA and 
      methylated DNA from formalin-fixed, paraffin-embedded specimens by quantitative 
      PCR.
PG  - 253-60
LID - S1525-1578(13)00257-2 [pii]
LID - 10.1016/j.jmoldx.2013.11.003 [doi]
AB  - Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from 
      formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in 
      clinical laboratory practice. Excluding specimens with poorly preserved nucleic 
      acids is an important quality-control step for avoiding unreliable results. 
      Because the assays for RNA abundance and DNA methylation have different critical 
      limiting factors, we examined the extent of overlap of excluded specimens for RNA 
      abundance versus methylated DNA. The transcript abundance of three reference 
      genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR 
      Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was 
      identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's 
      kappa. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to 
      determine usability in the MethyLight assay. Excluding specimens with mean 
      reference gene CT values exceeding the group mean by >1 SD led to significant 
      improvement of the concordance of ESR1 and ER. Specimens with usable DNA after 
      bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 
      SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA 
      extracted from the same FFPE block need separate exclusion criteria for qPCR 
      assays of transcript abundance and methylated DNA.
CI  - Copyright (c) 2014 American Society for Investigative Pathology and the Association 
      for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
FAU - Korlimarla, Aruna
AU  - Korlimarla A
AD  - Division of Molecular Medicine, St. John's Research Institute, Bangalore, India. 
      Electronic address: aruna@sjri.res.in.
FAU - Prabhu, Jyothi S
AU  - Prabhu JS
AD  - Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
FAU - Anupama, C E
AU  - Anupama CE
AD  - Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
FAU - Remacle, Jose
AU  - Remacle J
AD  - Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
FAU - Wahi, Kanu
AU  - Wahi K
AD  - Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
FAU - Sridhar, T S
AU  - Sridhar TS
AD  - Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20140109
PL  - United States
TA  - J Mol Diagn
JT  - The Journal of molecular diagnostics : JMD
JID - 100893612
RN  - 1HG84L3525 (Formaldehyde)
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Breast Neoplasms/diagnosis/genetics
MH  - DNA/*genetics/standards
MH  - *DNA Methylation
MH  - Female
MH  - Formaldehyde
MH  - Humans
MH  - Paraffin Embedding
MH  - Quality Control
MH  - RNA/*genetics/standards
MH  - Real-Time Polymerase Chain Reaction/*methods/*standards
MH  - Reproducibility of Results
MH  - Sensitivity and Specificity
MH  - Tissue Fixation
EDAT- 2014/01/15 06:00
MHDA- 2014/10/29 06:00
CRDT- 2014/01/14 06:00
PHST- 2013/08/31 00:00 [received]
PHST- 2013/11/13 00:00 [revised]
PHST- 2013/11/25 00:00 [accepted]
PHST- 2014/01/14 06:00 [entrez]
PHST- 2014/01/15 06:00 [pubmed]
PHST- 2014/10/29 06:00 [medline]
AID - S1525-1578(13)00257-2 [pii]
AID - 10.1016/j.jmoldx.2013.11.003 [doi]
PST - ppublish
SO  - J Mol Diagn. 2014 Mar;16(2):253-60. doi: 10.1016/j.jmoldx.2013.11.003. Epub 2014 
      Jan 9.