The selection of endogenous genes in human postmortem tissues.

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Accurate postmortem interval (PMI) determination is critical in forensic investigations.
• Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a valuable tool for analyzing gene expression in PMI studies.
• Data normalization using stable endogenous markers is necessary for reliable qRT-PCR results.

Purpose of the Study:

• To identify stable endogenous molecular markers for accurate PMI determination in human tissues.
• To evaluate the suitability of nine commonly used endogenous markers under varying postmortem conditions.
• To assess the influence of postmortem interval and cause of death on marker stability.

Main Methods:

• Comparison of nine candidate endogenous markers (ACTB, GAPDH, B2M, U6, 18S rRNA, hsa-mir-1, hsa-mir-9, hsa-mir-194-1, hsa-mir-203) in 109 human autopsy tissue samples.
• RNA extraction from heart, brain, kidney, and skin tissues.
• Gene expression analysis using quantitative RT-PCR (qRT-PCR) and assessment of marker stability with geNorm software.

Main Results:

• U6, GAPDH, and 18S rRNA were identified as suitable endogenous control genes across various postmortem conditions.
• ACTB and B2M demonstrated reliability as internal controls specifically in heart tissue.
• MicroRNAs (hsa-mir-1, hsa-mir-9, hsa-mir-194-1, hsa-mir-203) exhibited high M values, indicating unsuitability as endogenous controls.

Conclusions:

• U6, GAPDH, and 18S rRNA are recommended as stable endogenous markers for qRT-PCR-based PMI determination in diverse human tissues.
• The choice of endogenous control requires careful consideration of tissue type and experimental conditions.
• MicroRNAs are not recommended for use as endogenous controls in this context due to observed instability.