2.2 Validation of Autopsy and Specimen Collection Techniques and RNA Quality Assessment 2.2.1 Background As mentioned in the Chapter 1 that this D.Phil. project focuses on the development of molecular-based techniques to complement the understanding of pathogenesis of severe malaria gained by conventional histopathological and immunopathological approaches, this necessitates the access to human tissue with severe malaria infections and tissue of normal controls. In order to gain this access, our group has started an ongoing autopsy-based study in fatal malaria in Mozambique (details of this study will be discussed in the next section). Before the autopsy in Mozambique commenced, a study to validate the autopsy and specimen collection techniques, which would be used in Mozambique, had been done in Bangkok, Thailand. This study allowed us to perform a feasibility study of mRNA extraction and gene expression analysis on samples of post mortem human organs, optimising retrieval for molecular pathology studies by examining effects of pre- and post- mortem factors, tissue collection, storage and RNA isolation techniques. Moreover, this study form a part of tissue bank of control patients for the use in histological, immunohistochemical and the comparison of gene expression profile between malaria and non-malaria individuals
respiratory distress syndrome, ARF = acute renal failure, CA = cancer
RNA Sample Purity
The A260/A280 ratio and A260/A230 ratio are used to assess the sample purity. All
types of nucleic acid compounds including nucleotide, dsDNA and RNA have
absorbance at 260 nm, while protein, phenol, carbohydrates, EDTA, guanidine HCL,
guanidine isothiocyanate and other contaminants absorb at 280 or 230 nm. Generally
the A260/A280 ratio of ~2.0 is accepted as “pure” for RNA, and the A260/A230 is
commonly in the range of 2.0 – 2.2, which is often a little higher than A260/A280. If
the ratios are much lower than expected, it would indicate the presence of
Overall, RNA samples extracted from this series of autopsy are of high purity,
regardless of the differences in preservation techniques or the areas of the brain
(A260/A280 median = 2.08, IQR = 2.06 – 2.09; A260/A230 median = 2.14, IQR =
1.94 – 2.24). There was a significant difference in A260/A280 and A260/A230 ratios
between different areas of the brain (P =0.048, P =0.017, respectively). Medulla had
slightly lower RNA purity than the other areas, according to both ratios. However, the
preservation techniques did not affect the A260/A280 and A260/A230 ratios (P
=0.696, P =0.755, respectively) (Table Error! No text of specified style in document.-
Table Error! No text of specified style in document.-2 Summary of RNA purity measurements stratified by preservation techniques and areas of the brain
Overall 2.08 (2.06-2.09) 2.14 (1.94-2.24)
LNV 2.08 (2.06-2.08) 2.11 (1.85-2.16)
RNAlater A 2.08 (2.06-2.09) 2.17 (1.97-2.24)
RNAlater B 2.08 (2.06-2.09) 2.19 (1.99-2.24)
P-value* 0.696 0.755
Area of the brain
Cortex 2.08 (2.08-2.1) 2.15 (1.94-2.25)
Thalamus 2.08 (2.06-2.09) 2.21 (2.1-2.27)
Medulla 2.07 (2.06-2.08) 2.06 (1.83-2.2)
P-value* 0.048 0.017
*Kruskall-Wallis, Data presented as median (IRQ)
RNA Integrity Number (RIN)
Overall, RNA samples extracted from this series of autopsy are of medium quality,
according to RIN measured and calculated by Anglent 2100 Bioanalyzer (median =
5.7, IQR = 4.2 – 6.4). The RIN significantly differ between the areas of the brain (P =
0.024) and thalamus was likely to have lower RIN than the other areas. However the
preservation techniques did not affect the RIN (P = 0.301) (Table Error! No text of
specified style in document.-3).
It is noteworthy that when looked to into details in each case, thalamus did not have
the lowest RIN in every case and the patterns of RIN across different areas of the
brain were varied from case to case. For example, the pattern of RIN in one case
might be cortex > thalamus > medulla, but in another case the pattern might be
medulla > cortex >thalamus.
Table Error! No text of specified style in document.-3 Summary of RIN stratified by preservation techniques and areas of the brain
Overall 5.7 (4.2-6.4)
LNV 5.35 (3.8-6.3)
RNAlater A 6 (4.9-7)
RNAlater B 5.35 (4-6.4)
Area of the brain
Cortex 6.15 (5-7.3)
Thalamus 4.45 (3.9-6)
Medulla 6 (4.7-6.4)
*Kruskall-Wallis, Data presented as median (IQR)
Postmortem Interval between Death and Autopsy (PMI)
There were significantly negative correlations between postmortem interval and
A260/A230 ratio (Spearman’s rho = -0.262, P =0.012), and RIN (Spearman’s rho = -
0.375, P =0.002). No correlation with A260/A280 was found.
Interestingly, even with the shortest postmortem delay, the brain of case CU9 (4 hours
PMI) had relatively low averaged RIN (4.36), whilst the brain from case CU1 (5
hours PMI) had the highest averaged RIN (7.33). The examples of electropherogram
of some samples from CU1 and CU9 were presented in Figure Error! No text of
specified style in document.-1.
RNA extraction from the brain samples in this autopsy series using RNeasy Lipid
Tissue Mini Kit (Qiagen, UK) yielded high amount of total RNA (median 851 ng/ml,
IQR = 709 – 1093 ng/ml, total 60 ml). As expected, RNA extraction quantity was
significantly correlated with the amount of starting material (Spearman’s rho = 0.454,
P <0.000). However, there are unexpected significant correlations between the RNA
yield and A260/A280 ratio, A260/A230 ratio and RIN (Spearman’s rho = 0.321,
0.223, 0.264; P =0.002, 0.034, 0.033, respectively).
Figure Error! No text of specified style in document.-1 Digital gel-electrophoresis-like image and electropherograms of 6 representative RNA samples. (A) High quality RNA sample from tissue culture. (B – C) RNA samples from case CU1 which had PMI of 5 hours. These 2 samples had good quality of RNA according to RIN values; however, rRNA ratios of these samples were dramatically different. Sample C with rRNA ratio of 1.0 would have been traditionally considered as poor quality RNA sample. This evidence proved that the ratio of ribosomal bands is not reliably represent RNA degradation because this ratio takes only degradation of rRNA into account. (D-E) RNA from case CU9 which had PMI of 4 hours. This case was a good example of the existence of unpredictable and unexplainable confounds affecting RNA quality. Even with the similar pre- and post-mortem conditions as case CU1, this case (CU9) had much poorer RNA quality than case CU1. Moreover RIN value of thalamus was considerably lower than that of cortex of the same brain with the same preservation technique. (F) This RNA sample from case CU2 showed highly degraded RNA. This case had prolonged agonal status.
This study examined the quality of RNA samples extracted from human postmortem
tissues with the goal of validating autopsy procedure and specimen collection
techniques that would yield the best tissue quality for use in molecular study at
resource-limited setting. This study was considered as a feasibility study of an
ongoing autopsy study of fatal malaria in Mozambique and form a part of tissue bank
of control cases for use in the study of RNA expression profile and other molecular
studies in the future.
Data showed that storage of fresh human brain tissues in RNAlater at room
temperature of tropical country (27-35C) for 24 hours before storing in a normal
domestic freezer at -20C did not decrease the quality of RNA, compared to snap
freezing in vapor phase of liquid nitrogen which required much more specialised
procedures and equipments. This finding assured us the successful use of RNAlater as
a tissue preservative in Mozambique; the ambient room temperature is much higher in
tropical countries than temperate countries where most publication on RNAlater came
from. This finding enabled us to design a specimen collection protocol that is practical
for conducting research in resource-limited places such as Africa or in a decentralized
Postmortem interval was still one of the key factors determining tissue quality. It
significantly negatively correlated with RIN and A260/A280 ratio. This finding
suggested that the autopsy and tissue collection should be conducted as soon as
possible after death to minimise RNA degradation. However, tissue quality was not
only affected by post-mortem factors. Premortem agonal factors played a crucial in
tissue quality. This was supported by the finding that 2 cases with the shortest
postmortem delay (CU9 and CU1) had a significant difference in RNA degradation.
CU9, (PMI = 4hr), had averaged RIN of 4.36, while CU1, (PMI = 5hr), had averaged
RIN of 7.33. In addition, the case with the lowest RIN in this autopsy series is CU2,
who had prolonged agonal status before death. These indicated that patient source was
the important source of variation of the tissue quality.
Data also showed that all tested perseveration techniques were compatible with the
RNA isolation kit by Qiagen and they all yielded high quantity of high purity RNA.
This assured us that tissue in RNAlater was compatible with RNA isolation kit
designed of fresh frozen tissue and the quantity and quality of RNA were not affected
by different preservation techniques.
In conclusion, the main cause of variability in postmortem tissue quality was tissue
source (patients). Some factors could not be explained such as the variability of
quality in tissue from different area of the same brain or different persons with the
same agonal and post mortem statuses. Post mortem factors, especially post mortem
interval, also contributed to some extend to the RNA degradation, but most of these
factors can be controlled and manipulated by a well-designed study. Tissue in
RNAlater had a similar RNA quality to that of fresh frozen tissue and was compatible
with the similar RNA isolation kit. The use of RNAlater as an RNA preservative at
normal ambient temperature opens the opportunity to decentralised human tissue
collection in a resource-limited setting and later conduct molecular-based studies from
that human tissue in a well-equipped central laboratory.
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