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Analysis of microRNA from archived formalin-fixed paraffin-embedded specimens of amyotrophic lateral sclerosis.

Wakabayashi K, Mori F, Kakita A, Takahashi H, Utsumi J, Sasaki H - Acta Neuropathol Commun (2014)

Bottom Line: An overall RNA extraction success rate was 46.2%.Target genes were predicted using miRNA bioinformatics software, and the data applied to ontology analysis.This indicated that one of the miRNAs up-regulated in ALS (miR-338-3p) had already been identified in leukocytes, serum, cerebrospinal fluid and frozen spinal cord from ALS patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan. koichi@cc.hirosaki-u.ac.jp.

ABSTRACT

Background: MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression. This study investigated whether formalin-fixed paraffin-embedded (FFPE) specimens from postmortem cases of neurodegenerative disorders would be suitable for miRNA profiling.

Results: Ten FFPE samples from 6 cases of amyotrophic lateral sclerosis (ALS) and 4 neurologically normal controls were selected for miRNA analysis on the basis of the following criteria for RNA quality: (i) a postmortem interval of less than 6 hours, (ii) a formalin fixation time of less than 4 weeks, (iii) an RNA yield per sample of more than 500 ng, and (iv) sufficient quality of the RNA agarose gel image. An overall RNA extraction success rate was 46.2%. For ALS, a total of 364 miRNAs were identified in the motor cortex, 91 being up-regulated and 233 down-regulated. Target genes were predicted using miRNA bioinformatics software, and the data applied to ontology analysis. This indicated that one of the miRNAs up-regulated in ALS (miR-338-3p) had already been identified in leukocytes, serum, cerebrospinal fluid and frozen spinal cord from ALS patients.

Conclusion: Although analysis was possible for just under half of the specimens examined, we were able to show that informative miRNA data can be derived from archived FFPE samples from postmortem cases of neurodegenerative disorders.

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Related in: MedlinePlus

Effect of formalin fixation on RNA expression. Representative RNA agarose gel image showing a band of 2000 nt corresponding to 18S ribosomal RNA in lane 1. A faint band of 2000 nt is also seen in lanes 2 and 3. Lane 1, sample 2; lane 2, sample 1; lane 3, sample 8.
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Fig1: Effect of formalin fixation on RNA expression. Representative RNA agarose gel image showing a band of 2000 nt corresponding to 18S ribosomal RNA in lane 1. A faint band of 2000 nt is also seen in lanes 2 and 3. Lane 1, sample 2; lane 2, sample 1; lane 3, sample 8.

Mentions: The RNA yield was not correlated with the period of storage of FFPE blocks. However, the RNA yield was influenced by the period of formalin fixation (Table 1), being significantly higher in samples that had been fixed for a short period (3–4 weeks; mean 2465 ng) than in those that have been fixed for a long period (8–16 weeks; mean 574 ng) (p < 0.05). The RNA yield did not appear to be affected by the type of fixative employed. Postmortem interval could be also relevant. Samples with higher RNA yield (samples 1, 2 and 8) were the cases in whom postmortem interval was within 4 hours. The rest with lower RNA yield (samples 3–7, 9 and 10) had a combination of longer postmortem interval (9–10 hours) and/or fixation (8–16 weeks). RNA integrity was checked by electrophoresis, and a representative RNA agarose gel image is shown in Figure 1. A band of 5000 nt corresponding to 28S ribosomal RNA was slightly shifted toward a higher molecular weight in all cases, indicating overfixation with formalin and the presence of chemical modifications of the RNA such as covalently linked residual amino acids [59]. A band of 2000 nt corresponding to 18S ribosomal RNA was seen in some cases (Figure 1), suggesting that the extracted RNA was of good quality. These bands were not only shifted, but also appeared more diffuse and less focused. The bands of 100–200 nt corresponded to degraded RNA, as reported previously [60]. On the basis of these initial analyses, we adopted four criteria to indicate that RNA would be of sufficient quality for analysis, based on Osawa et al. [50]: (i) a postmortem interval of less than 6 hours, (ii) a formalin fixation time of less than 4 weeks, (iii) a total RNA yield per sample of more than 500 ng, and (iv) a RNA electrophoresis pattern of good quality.Figure 1


Analysis of microRNA from archived formalin-fixed paraffin-embedded specimens of amyotrophic lateral sclerosis.

Wakabayashi K, Mori F, Kakita A, Takahashi H, Utsumi J, Sasaki H - Acta Neuropathol Commun (2014)

Effect of formalin fixation on RNA expression. Representative RNA agarose gel image showing a band of 2000 nt corresponding to 18S ribosomal RNA in lane 1. A faint band of 2000 nt is also seen in lanes 2 and 3. Lane 1, sample 2; lane 2, sample 1; lane 3, sample 8.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279903&req=5

Fig1: Effect of formalin fixation on RNA expression. Representative RNA agarose gel image showing a band of 2000 nt corresponding to 18S ribosomal RNA in lane 1. A faint band of 2000 nt is also seen in lanes 2 and 3. Lane 1, sample 2; lane 2, sample 1; lane 3, sample 8.
Mentions: The RNA yield was not correlated with the period of storage of FFPE blocks. However, the RNA yield was influenced by the period of formalin fixation (Table 1), being significantly higher in samples that had been fixed for a short period (3–4 weeks; mean 2465 ng) than in those that have been fixed for a long period (8–16 weeks; mean 574 ng) (p < 0.05). The RNA yield did not appear to be affected by the type of fixative employed. Postmortem interval could be also relevant. Samples with higher RNA yield (samples 1, 2 and 8) were the cases in whom postmortem interval was within 4 hours. The rest with lower RNA yield (samples 3–7, 9 and 10) had a combination of longer postmortem interval (9–10 hours) and/or fixation (8–16 weeks). RNA integrity was checked by electrophoresis, and a representative RNA agarose gel image is shown in Figure 1. A band of 5000 nt corresponding to 28S ribosomal RNA was slightly shifted toward a higher molecular weight in all cases, indicating overfixation with formalin and the presence of chemical modifications of the RNA such as covalently linked residual amino acids [59]. A band of 2000 nt corresponding to 18S ribosomal RNA was seen in some cases (Figure 1), suggesting that the extracted RNA was of good quality. These bands were not only shifted, but also appeared more diffuse and less focused. The bands of 100–200 nt corresponded to degraded RNA, as reported previously [60]. On the basis of these initial analyses, we adopted four criteria to indicate that RNA would be of sufficient quality for analysis, based on Osawa et al. [50]: (i) a postmortem interval of less than 6 hours, (ii) a formalin fixation time of less than 4 weeks, (iii) a total RNA yield per sample of more than 500 ng, and (iv) a RNA electrophoresis pattern of good quality.Figure 1

Bottom Line: An overall RNA extraction success rate was 46.2%.Target genes were predicted using miRNA bioinformatics software, and the data applied to ontology analysis.This indicated that one of the miRNAs up-regulated in ALS (miR-338-3p) had already been identified in leukocytes, serum, cerebrospinal fluid and frozen spinal cord from ALS patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan. koichi@cc.hirosaki-u.ac.jp.

ABSTRACT

Background: MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression. This study investigated whether formalin-fixed paraffin-embedded (FFPE) specimens from postmortem cases of neurodegenerative disorders would be suitable for miRNA profiling.

Results: Ten FFPE samples from 6 cases of amyotrophic lateral sclerosis (ALS) and 4 neurologically normal controls were selected for miRNA analysis on the basis of the following criteria for RNA quality: (i) a postmortem interval of less than 6 hours, (ii) a formalin fixation time of less than 4 weeks, (iii) an RNA yield per sample of more than 500 ng, and (iv) sufficient quality of the RNA agarose gel image. An overall RNA extraction success rate was 46.2%. For ALS, a total of 364 miRNAs were identified in the motor cortex, 91 being up-regulated and 233 down-regulated. Target genes were predicted using miRNA bioinformatics software, and the data applied to ontology analysis. This indicated that one of the miRNAs up-regulated in ALS (miR-338-3p) had already been identified in leukocytes, serum, cerebrospinal fluid and frozen spinal cord from ALS patients.

Conclusion: Although analysis was possible for just under half of the specimens examined, we were able to show that informative miRNA data can be derived from archived FFPE samples from postmortem cases of neurodegenerative disorders.

Show MeSH
Related in: MedlinePlus