Validation of adequate endogenous reference genes for the normalisation of qPCR gene expression data in human post mortem tissue.

Author(s): Koppelkamm A, Vennemann B, Fracasso T, Lutz-Bonengel S, Schmidt U, Heinrich M

Publication: Int J Legal Med, 2010, Vol. 124, Page 371-80

PubMed ID: 20300940 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the stability of ten common real-time qRT-PCR reference genes (ACTB, 18S rRNA, GAPDH, TBP, HPRT1, TBP, HPRT1, B2M, HMBS, SDHA, CYCA, UBC) in specimens that varied with regard to age at time of death, cause of death, BMI, gender, and PMI. Cardiac muscle, skeletal muscle, and brain specimens were collected from 37 patients during autopsy and preserved in RNAlater prior to storage at -80 degrees C.

   Summary of Findings:

   RNA yield was influenced by tissue type, with the greatest yield isolated from cardiac muscle, although no correlation to cause of death, age at death, or PMI was observed. Transcript stability was also tissue-dependent, as CYCA and TBP were the most stable transcripts in cardiac muscle, while SDHA, TBP, HPRT1, and CYCA were the most stable in skeletal muscle, and HMBS, SDHA, GAPDH, and UBC were the most stable in brain. The authors recommend a minimum of 5 or 6 reference genes for real-time qRT-PCR expression analysis in cardiac and skeletal muscle or brain, respectively.

   Biospecimens

   • Tissue - Heart
   • Tissue - Brain
   • Tissue - Muscle (Skeletal)

   Preservative Types

   • RNAlater

   Diagnoses:

   • Autopsy
   • Not specified

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Preaquisition	Cause of death	Other (not specified) Central dysregulation Sudden cardiac death Asphyxia
   Biospecimen Acquisition	Biospecimen location	Cardiac muscle Skeletal muscle Brain
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB 18S rRNA GAPDH TBP HPRT1 B2M HMBS SDHA CYCA UBC
   Preaquisition	Patient body mass index	16.4-37.7
   Preaquisition	Patient age	12-87 y
   Preaquisition	Postmortem interval	4-42 h

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2. Study Purpose

   The purpose of this study was to determine if age at time of death, cause of death, BMI, gender, or PMI was associated with significant differences in transcript levels of six common real-time qRT-PCR reference genes (ACTB, 18S rRNA, GAPDH, HPRT1, B2M, UBC). Each preanalytical variable was assessed by differential segregation of a common and diverse sample set. Specimens were collected from a total of 37 individuals at autopsy, although sample size per group ranged between 7 and 23 individuals. Skeletal muscle, cardiac muscle, and brain specimens were preserved in RNAlater prior to storage at -80 degrees C.

   Summary of Findings:

   Significant fluctuations in transcript levels were associated with each preanalytical variable investigated, although differences were gene- and tissue-specific. While all six transcripts investigated were stable in brain specimens exposed to a PMI of 30 h, in skeletal muscle GAPDH levels were significantly higher after a PMI of 11-20 h or longer compared to 0-10 h controls, and UBC levels were significantly depressed after a PMI of 21-30 h compared to a shorter or longer PMI. Interestingly, in cardiac muscle GAPDH levels were significantly lower after a PMI of 21-30 h while B2M levels were significantly higher in comparison to 0-10 h controls. Cause of death affected transcript levels in skeletal muscle and brain, but not cardiac muscle; B2M and GAPDH levels were higher in skeletal muscle specimens following death from a sudden cardiac event or asphyxia, respectively, while B2M, HPRT1, and TBP were higher in brain specimens following central dysregulation compared to other causes of death. The only significant difference between genders was elevated UBC levels in skeletal muscle collected from females compared to males. Significant effects of age at time of death were limited to higher GAPDH and 18S rRNA levels in skeletal muscle collected from individuals that were 30 years of age or younger compared to older individuals. A BMI of 25 or greater at death was associated with significantly higher ACTB and lower GAPDH levels in skeletal muscle compared to lower BMI.

   Biospecimens

   • Tissue - Heart
   • Tissue - Muscle (Skeletal)
   • Tissue - Brain

   Preservative Types

   • RNAlater

   Diagnoses:

   • Autopsy
   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	<30 y 31-50 y >50 y
   Preaquisition	Patient gender	Female Male
   Preaquisition	Cause of death	Other (not specified) Central dysregulation Sudden cardiac death Asphyxia
   Preaquisition	Postmortem interval	<10 h 11-20 h 21-30 h 31-45 h
   Biospecimen Acquisition	Biospecimen location	Cardiac muscle Skeletal muscle Brain
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB 18S rRNA GAPDH HPRT1 B2M UBC
   Preaquisition	Patient body mass index	<25 >25

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