RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays.
Author(s): Koppelkamm A, Vennemann B, Lutz-Bonengel S, Fracasso T, Vennemann M
Publication: Int J Legal Med, 2011, Vol. 125, Page 573-80
PubMed ID: 21584656 PubMed Review Paper? No
Purpose of Paper
The purpose of the paper was to identify the effect of tissue type, age at death, gender, body mass index (BMI), duration of agony, cause of death and post-mortem interval (PMI) on RNA integrity numbers (RIN) and to analyze the influence of RNA integrity and normalization on data obtained with real-time qRT-PCR.
Conclusion of Paper
RNA extracted from post-mortem brain specimens had significantly lower RIN values than that from skeletal muscle (mean RIN of 2.8 versus 4.4) and cardiac muscle (mean RIN of 2.8 versus 3.8). BMI was found to have a statistically significant influence on RIN in skeletal muscle , but age of the deceased, gender, duration of agony and cause of death had no statistically significant influence in any of the tissue types. RIN numbers in experimentally degraded RNA demonstrated strong to very strong correlations with cycle of quantification values (Cq) of each of the three housekeeping genes, but the magnitude of the effect depended on the transcript and whether the RNA was obtained from heart, brain or skeletal muscle. Normalization with calibrated normalized relative quantities (CNRQ) eliminated the effect of RIN on B2M levels, but the authors state the effects of RIN on ACTB and 18S rRNA levels were not consistent between studies and thus the effect of CNRQ normalization was not elucidated.
The purpose of this study was to determine if type of tissue, gender, body mass index (BMI), age at death, cause of death, duration of agony, or post-mortem interval (PMI) influence RIN values and the reliability of quantitative gene expression data in human brain, cardiac muscle and skeletal muscle tissues and to investigate if using a normalization method on real-time-PCR data could reduce the influence of impaired RNA integrity. A total of 136 samples (45 brain frontal lobe samples, 47 cardiac muscle samples, 44 skeletal muscle samples) without obvious pathological changes from 49 individuals were collected during forensic routine autopsies in RNAlater and transferred to −80°C for long time storage. RNA was isolated from post-mortem tissue samples using RNagents Total RNA Isolation System and phenol/chloroform/isoamyl alcohol. RNA integrity was assessed by bioanalyzer and RIN values were assigned ranging from 1 (highly degraded) to 10 (intact) total RNA. To investigate the effect of RNA degradation on real-time qRT-PCR results directly, commercially available total RNA from human brain, heart and skeletal muscle was degraded by incubation at 70°C for 10 different times ranging from 0 to 175 min and immediately transferred to ice.
Summary of Findings:
RNA extracted from post-mortem brain specimens had significantly lower RIN values than that from skeletal muscle (mean RIN of 2.8 versus 4.4, p<0.05) and cardiac muscle (mean RIN of 2.8 versus 3.8, p<0.05). A weak, but statistically significant correlation between RIN and PMI was observed in post-mortem cardiac muscle tissue (R=-0.347, p<0.05), but due to a high degree of variation in the data points, it is unclear if this is due to PMI influence alone. RIN of RNA extracted from the skeletal muscle of obese donors (BMI> 25) was significantly lower compared to samples from normal weight donors, but no effect of BMI on RIN of RNA from brain or cardiac muscle was observed. The authors attribute this effect to anatomical location and the rate of post-mortem cooling. Age of the deceased, gender, agonal duration and cause of death did not have a statistically significant influence on RIN in any of the tissue types. Statistically significant strong to very strong correlation coefficients between Cq for each of the three transcripts (18S RNA, ACTB, and B2M) and RIN values were found for experimentally degraded RNA from each of the three tissues. However, degradation resulted in a 7-fold change in the quantified levels of18S rRNA from heart, but only an approximately 2-fold change in ACTB levels was observed, regardless of tissue. Thus the authors conclude that the effect of RNA integrity on Cq is dependent on the transcript analyzed as well as the tissue from which the RNA was obtained. A statistically significant correlation between RIN and non-normalized Cq (NN-Cq) was observed for B2M in cardiac muscle tissue (p<0.01), but this effect was eliminated by CNRQ. While the authors report a strong correlation between RIN and NN-Cq was observed in the degradation curve for ACTB and 18S rRNA in cDNA generated from commercially available RNA, they were unable to confirm in post-mortem specimens and did not discuss the effect of normalization by CNRQ.
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Preaquisition Postmortem interval 5 - 44 hrs
Preaquisition Patient body mass index >25 (pre-obese or obese)
Preaquisition Patient age <35 years
35 - 50 years
Preaquisition Cause of death suffocation (deaths due to mechanical airway obstruction and external chest compression)
central dysregulation (deaths due to CNS depression such as in opiate overdose)
sudden cardiac death (deaths due to acute myocardial ischemia and infarction)
bleeding to death (fatal internal or external hemorrhage due to traumatic or natural causes)
Real-time qRT-PCR Specific Targeted nucleic acid ACTB