Intra- and Inter-individual Variability of microRNA Levels in Human Cerebrospinal Fluid: Critical Implications for Biomarker Discovery

Stochastic dynamics have a pronounced effect on the level of molecules especially when the level of the molecule is low.

“Such stochastic dynamics will have pronounced effect on the level of molecules especially when the level of molecule is low24.”

• cites [24] Toni, T. & Tidor, B. Combined model of intrinsic and extrinsic variability for computational network design with application to synthetic biology. PLoS Comput Biol 9, e1002960, https://doi.org/10.1371/journal.pcbi.1002960 (2013). sourceSupported

There has been a lack of consistent findings among studies of microRNAs in cerebrospinal fluid.

“However, there has been a lack of consistent findings among CSF microRNAs studies.”

• Also stated in: § Introduction · plain

  “Although several studies have identified the differentially expressed miRNAs in the CSF of AD patients, the miRNA profiling results were not well replicated from one cohort to another.”

• cites [11] Alexandrov, P. N. et al. microRNA (miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF). Int J Biochem Mol Biol 3, 365–373 (2012).Not checked — no full text of the source
• cites [12] Denk, J. et al. MicroRNA Profiling of CSF Reveals Potential Biomarkers to Detect Alzheimer’s Disease. PLoS One 10, e0126423, https://doi.org/10.1371/journal.pone.0126423 (2015). sourceSupported
• cites [13] Kiko, T. et al. MicroRNAs in plasma and cerebrospinal fluid as potential markers for Alzheimer’s disease. J Alzheimers Dis 39, 253–259, https://doi.org/10.3233/JAD-130932 (2014). sourceNot checked — no full text of the source

Inconsistent results among CSF microRNA studies have been attributed in part to various technical limitations and demographic diversity between sample cohorts.

“Obviously, various technical limitations and demographic diversity between sample cohorts may have contributed, in part, to such conflicting results20–22.”

• cites [20] Pritchard, C. C., Cheng, H. H. & Tewari, M. MicroRNA profiling: approaches and considerations. Nat Rev Genet 13, 358–369, https://doi.org/10.1038/nrg3198 (2012). sourcePartly supported

  The Data Analysis and Interpretation section describes technical biases and batch effects in miRNA profiling, while Conclusions and Perspective notes sample-to-sample biological variability. The paper does not attribute conflicting CSF study results specifically to those factors or identify demographic diversity between cohorts as a cause. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [21] Moldovan, L. et al. Methodological challenges in utilizing miRNAs as circulating biomarkers. J Cell Mol Med 18, 371–390, https://doi.org/10.1111/jcmm.12236 (2014). sourcePartly supported

  The Introduction (page unavailable) says differences in extraction methods and analysis platforms can produce inconsistent or contradictory miRNA results. The paper focuses on plasma and serum; our copy does not attribute conflicting CSF study results to those limitations or to demographic differences between cohorts. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [22] van Harten, A. C., Mulders, J., Scheltens, P., van der Flier, W. M. & Oudejans, C. B. Differential Expression of microRNA in Cerebrospinal Fluid as a Potential Novel Biomarker for Alzheimer’s Disease. J Alzheimers Dis 47, 243–252, https://doi.org/10.3233/JAD-140075 (2015). sourceSupported

More miRNAs can be detected in postmortem CSF because CSF can be contaminated by miRNAs released from degenerated brain tissues.

“It has been reported that more miRNAs can be detected in postmortem CSF, because CSF can be contaminated by miRNAs released from degenerated brain tissues19,37.”

• cites [19] Sala Frigerio, C. et al. Reduced expression of hsa-miR-27a-3p in CSF of patients with Alzheimer disease. Neurology 81, 2103–2106, https://doi.org/10.1212/01.wnl.0000437306.37850.22 (2013). sourceNot checked — no full text of the source
• cites [37] Barbato, C. Alzheim(i)R: MicroRNAs in Alzheimer’s Disease. Journal of Cytology & Histology 05, https://doi.org/10.4172/2157-7099.1000e113 (2014). sourceSupported

About 100 miRNAs account for most miRNAs in the brain.

“Because miRNAs have strong tissue-specific expression patterns, only approximately 300 miRNAs out of ~2,500 total human miRNAs were detected in human brain tissues, and about 100 miRNAs account for most miRNAs in the brain38,39.”

• cites [38] Shin, C. et al. Expanding the microRNA targeting code: functional sites with centered pairing. Mol Cell 38, 789–802, https://doi.org/10.1016/j.molcel.2010.06.005 (2010). sourceCould not verify

  The Results section, “MicroRNA-directed Cleavage in HeLa Cells and Human brain” (page unavailable), says brain-expressed miRNAs were identified by small-RNA sequencing, but gives no count or distribution of their abundance. Searches for approximately 100 miRNAs and the most abundant brain miRNAs found no passage supporting the claim that about 100 account for most brain miRNAs. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [39] Hu, H. Y. et al. MicroRNA expression and regulation in human, chimpanzee, and macaque brains. PLoS Genet 7, e1002327, https://doi.org/10.1371/journal.pgen.1002327 (2011). sourceCould not verify

  The Results/Discussion section on high-throughput sequencing reports 413 miRNAs detected in human prefrontal cortex or cerebellum, but does not say how many account for most brain miRNA expression. Searches for “top 100 miRNA” and for miRNA abundance likewise found no passage supporting the claim about approximately 100 miRNAs. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

There has been a lack of consistent findings and even conflicting data among miRNA biomarker studies.

“Although the expression profiles of circulating miRNAs have been extensively studied under various disease conditions, it is noteworthy that there has been a lack of consistent finding and even conflicting data among miRNA biomarker studies16,20–22.”

• cites [16] Muller, M. et al. Validation of microRNAs in Cerebrospinal Fluid as Biomarkers for Different Forms of Dementia in a Multicenter Study. J Alzheimers Dis 52, 1321–1333, https://doi.org/10.3233/JAD-160038 (2016). sourceNot checked — no full text of the source
• cites [20] Pritchard, C. C., Cheng, H. H. & Tewari, M. MicroRNA profiling: approaches and considerations. Nat Rev Genet 13, 358–369, https://doi.org/10.1038/nrg3198 (2012). sourceCould not verify

  The Circulating Biomarkers section (page unavailable) says circulating miRNAs have been studied as disease biomarkers and raises concern about controlling pre-analytic and analytic variables. It does not say that biomarker studies have produced inconsistent or conflicting findings; searches for discordance and reproducibility did not locate that claim in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [21] Moldovan, L. et al. Methodological challenges in utilizing miRNAs as circulating biomarkers. J Cell Mol Med 18, 371–390, https://doi.org/10.1111/jcmm.12236 (2014). sourceSupported
• cites [22] van Harten, A. C., Mulders, J., Scheltens, P., van der Flier, W. M. & Oudejans, C. B. Differential Expression of microRNA in Cerebrospinal Fluid as a Potential Novel Biomarker for Alzheimer’s Disease. J Alzheimers Dis 47, 243–252, https://doi.org/10.3233/JAD-140075 (2015). sourcePartly supported

  The Discussion (page not provided) reports discrepancies between this study’s initial and validation results, and a finding in a prior Alzheimer’s CSF study that this study did not reproduce. This supports inconsistency in that specific area, but not the broader claim about miRNA biomarker studies across various diseases and circulating samples. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Approximately 300 out of ~2,500 total human miRNAs were detected in human brain tissues.

“Because miRNAs have strong tissue-specific expression patterns, only approximately 300 miRNAs out of ~2,500 total human miRNAs were detected in human brain tissues, and about 100 miRNAs account for most miRNAs in the brain38,39.”

• cites [38] Shin, C. et al. Expanding the microRNA targeting code: functional sites with centered pairing. Mol Cell 38, 789–802, https://doi.org/10.1016/j.molcel.2010.06.005 (2010). sourcePartly supported

  The Results section, “MicroRNA-directed Cleavage in HeLa Cells and Human brain” (page not available), says that miRNAs expressed in human brain tissue were identified by small-RNA sequencing. It does not give the claimed count of approximately 300 or a total of approximately 2,500 human miRNAs; searches for those numbers found no supporting passage. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [39] Hu, H. Y. et al. MicroRNA expression and regulation in human, chimpanzee, and macaque brains. PLoS Genet 7, e1002327, https://doi.org/10.1371/journal.pgen.1002327 (2011). sourcePartly supported

  The Materials and Methods section refers to 325 human miRNAs expressed in the brain, which supports the approximate figure. However, the Results/Discussion section reports detecting 413 miRNAs in the human prefrontal cortex or cerebellum, and the cited passages do not establish a total of approximately 2,500 human miRNAs. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The discrepancy between CSF biomarker studies may be in part due to the intrinsic variability of miRNA itself.

“Taken together, our data suggests that the discrepancy between CSF biomarker studies may be in part due to the intrinsic variability of miRNA itself43,44.”

• cites [43] Stylli, S. S. et al. miRNA expression profiling of cerebrospinal fluid in patients with aneurysmal subarachnoid hemorrhage. J Neurosurg 126, 1131–1139, https://doi.org/10.3171/2016.1.JNS151454 (2017). sourceCould not verify

  The Methods and Results (page unavailable) describe CSF sampling at different times and miRNA differences between patient groups. The Conclusions discuss the potential for CSF biomarkers, but no retrieved passage addresses intrinsic miRNA variability as an explanation for discrepancies between biomarker studies. I searched for intrinsic variability, study discrepancies, and temporal changes in miRNA expression. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [44] Marques, T. M. et al. MicroRNAs in Cerebrospinal Fluid as Potential Biomarkers for Parkinson’s Disease and Multiple System Atrophy. Mol Neurobiol. https://doi.org/10.1007/s12035-016-0253-0 (2016). sourcePartly supported

  The Discussion’s Study Limitations section (page not provided) says CSF miRNA measurements can vary across studies, citing sample processing, normalization methods, and qPCR variability. It does not identify intrinsic variability of miRNA itself as the cause of those discrepancies. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: Inherent physiological characteristics, such as noise in miRNA gene expression over a short period, may be responsible for conflicting results among miRNA biomarker studies.

miRNAs have strong tissue-specific expression patterns.

“Because miRNAs have strong tissue-specific expression patterns, only approximately 300 miRNAs out of ~2,500 total human miRNAs were detected in human brain tissues, and about 100 miRNAs account for most miRNAs in the brain38,39.”

• cites [38] Shin, C. et al. Expanding the microRNA targeting code: functional sites with centered pairing. Mol Cell 38, 789–802, https://doi.org/10.1016/j.molcel.2010.06.005 (2010). sourcePartly supported

  The Discussion section (page unavailable) attributes the small overlap in cleavage targets between human brain and HeLa cells partly to differential expression of miRNAs or mRNAs. That supports expression differences between these samples, but it does not establish strong tissue-specific expression patterns for miRNAs generally. The passages also do not support the cited sentence’s counts of approximately 300 brain miRNAs or 100 accounting for most brain miRNAs. (LLM, conf=0.77)

  Experimental — accuracy not yet measured
• cites [39] Hu, H. Y. et al. MicroRNA expression and regulation in human, chimpanzee, and macaque brains. PLoS Genet 7, e1002327, https://doi.org/10.1371/journal.pgen.1002327 (2011). sourceCould not verify

  The Materials and Methods section reports 325 human miRNAs expressed in brain, but it does not compare their expression across tissues. Searches for tissue-specific miRNA expression found no passage in our copy establishing the claimed strong tissue-specific patterns. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

83 miRNAs could be reliably detected in human CSF.

“As a result, we found that 83 miRNAs could be reliably detected in human CSF (Supplementary Table S1).”

miR-1246 and miR-374b-5p were identified as the two miRNAs with the lowest variability across all samples without considering the designated groups.

“miR-1246 and miR-374b-5p were identified as the two miRNAs with the lowest variability across all samples without considering the designated groups (Fig. 2B).”

All individuals except subject #5 were grouped together in the PCA plot of miRNA expression patterns.

“All individuals were grouped together in the plot, except the subject #5.”

miR-1246 and miR-374b-5p were also identified as the most consistent miRNAs within the designated groups.

“The same two miRNAs were also identified as the most consistent miRNAs within the designated groups (Fig. 2B).”

miR-1246 and miR-374b-5p exhibited similar expression patterns with small Ct differences between 0 and 48 hours.

“Both miRNAs exhibited a similar expression pattern with small Ct differences between 0 and 48 hours (Fig. 2C and D).”

The levels of most microRNAs in CSF were stable between 0 and 48 hours in healthy individuals.

“Surprisingly, the levels of most microRNAs were stable between the two time points.”

• Also stated in: § Introduction · plain

  “Surprisingly, we found that the levels of most miRNAs in CSF are not variable during 48 hours interval.”

The levels of 12 microRNAs (miR-19a-3p, miR-19b-3p, miR-23a-3p, miR-25a-3p, miR-99a-5p, miR-101-3p, miR-125b-5p, miR-130a-3p, miR-194-5p, miR-195-5p, miR-223-3p, and miR-451a) were significantly altered during the 48-hour interval in healthy individuals.

“However, the levels of 12 microRNAs (miR-19a-3p, miR-19b-3p, miR-23a-3p, miR-25a-3p, miR-99a-5p, miR-101-3p, miR-125b-5p, miR-130a-3p, miR-194-5p, miR-195-5p, miR-223-3p, and miR-451a) were significantly altered during the 48 hours interval.”

• Also stated in: § Introduction · plain

  “We also identified a few miRNAs whose expression levels were altered during the 48 hours.”

The R2 values from subject #1 to #9 for Ct-Ct correlation were 0.98, 0.98, 0.99, 0.99, 0.91, 0.98, 0.97, 0.99, and 0.99, respectively.

“The R 2 values from subject #1 to #9 were 0.98, 0.98, 0.99, 0.99, 0.91, 0.98, 0.97, 0.99, and 0.99, respectively.”

miR-125b-5p and miR-101-3p showed dramatically variable expression patterns during initial miRNA expression profiling.

“During initial miRNA expression profiling, miR-125b-5p and miR-101-3p showed dramatically variable expression patterns (Fig. 7A and B).”

We detected a relatively smaller number of miRNAs in CSF compared to other miRNA profiling studies.

“We detected a relatively smaller number of miRNAs in CSF, compared to other miRNA profiling studies19,28.”

• cites [19] Sala Frigerio, C. et al. Reduced expression of hsa-miR-27a-3p in CSF of patients with Alzheimer disease. Neurology 81, 2103–2106, https://doi.org/10.1212/01.wnl.0000437306.37850.22 (2013). sourceNot checked — no full text of the source
• cites [28] Cogswell, J. P. et al. Identification of miRNA changes in Alzheimer’s disease brain and CSF yields putative biomarkers and insights into disease pathways. J Alzheimers Dis 14, 27–41 (2008). sourceNot checked — no full text of the source

In the PCA plot of miRNAs, approximately 10 miRNAs were dispersed from the central cluster.

“A relatively tight clustering of miRNA data points was observed in the center of the PCA plot, while approximately 10 miRNAs were dispersed from the cluster.”

The levels of miR-125b were highly variable even within the same individual during the 48-hour interval in almost 90% of tested participants.

“In our experiments, the levels of miR-125b were highly variable even within the same individual during the 48-hour interval in almost 90% of tested participants (Fig. 4J).”

Removing subject #5 dramatically decreased the number of outlier miRNAs in the PCA plot.

“As shown in Fig. 3D, removing the subject #5 dramatically decreased the number of outlier miRNAs.”

Based on the 95% CI criteria, multiple miRNAs had significantly variable expression in more than four individuals.

“Based on the 95% CI criteria, we identified multiple miRNAs that had significantly variable expression in more than four individuals (Fig. 6C).”

In addition to the variable miRNAs listed in Fig. 4, the expression levels of miR-19b-3p, miR-23a-3p, and miR-99a-5p were also variable by using the SD of log2 RQV.

“In addition to the variable miRNAs listed in Fig. 4, we found that the expression levels of miR-19b-3p, miR-23a-3p, and miR-99a-5p were also variable by using the SD of log2 RQV.”

The stable miRNAs selected from the center of the PCA cluster exhibited very low intra- and inter-individual variability across all individuals during the 48-hour interval.

“These miRNAs exhibited very low intra- and inter-individual variability across all individuals during the 48-hour interval.”

The log2 RQVs of miR-223-3p varied among participants with variable effect size and direction.

“The log2 RQVs of miR-223-3p varied among participants with variable effect size and direction.”

By using Ct-Ct correlation analysis, miR-25-3p, miR-130a-3p, and miR-194-5p were additionally identified as intra-individually variable miRNAs.

“By using this Ct-Ct correlation analysis, miR-25-3p, miR-130a-3p, and miR-194-5p were additionally identified as intra-individually variable miRNAs.”

Subject #5 showed dramatic variability in multiple miRNA expression levels compared to other subjects.

“As expected from the PCA analysis, the subject #5 showed dramatic variability in the multiple miRNA expression levels compared to the other subjects.”

There was a strong correlation between the two independent replication experiments for 12 miRNAs with highly variable or stable expression.

“Using the same linear regression analysis method, we found that there was a strong correlation between the two independent replication experiments (Fig. 7G).”

No significant correlation was found between Ct values and miRNA variability when variability was assessed by the SD of log2 RQV.

“No significant correlation was found between the Ct values and the miRNA variability, when variability was assessed by the SD of log2 RQV (Fig. 7H).”

When the experiments were later repeated, almost identical data were reproduced.

“When the experiments were later repeated, we reproduced almost identical data (Fig. 7C and D).”

The SD reflecting inter-individual variability decreased dramatically when subject #5 was excluded.

“The SD reflecting inter-individual variability decreased dramatically when the subject #5 was excluded.”

Several miRNAs showed short-term intra- or inter-individual variability in their expression levels.

“we identified several miRNAs that showed short term intra- or inter-individual variability in their expression levels.”

Twelve miRNAs had high intra- or inter-individual variability in human CSF.

“However, we identified 12 miRNAs that had high intra- or inter-individual variability.”

• Also stated in: § Discussion · plain

  “While most miRNAs were very stable, we also identified 12 miRNAs with a high variability in human CSF (Table 1).”

• Also stated in: § Validation of technical reliability in miRNA measurement · plain

  “we identified 12 miRNAs with high intra- or inter-individual variability.”

miR-195-5p, miR-125b-5p, miR-101-3p, and miR-19a-3p exhibited a consistent decrease in their levels at 48 hours compared to 0 hours across most individuals.

“Conversely, miR-195-5p, miR-125b-5p, miR-101-3p, and miR-19a-3p exhibited a consistent decrease in their levels at the 48-hour time point compared to the 0-hour time point across most individuals (Fig. 4I–L).”

The initial screening and the validated qRT-PCR data were significantly correlated by linear regression analysis.

“The linear regression analysis indicated that the initial screening and the validated qRT-PCR data were significantly correlated (Fig. 7E and F).”

miR-451a was classified as a variable miRNA in the PCA plot.

“miR-451a was also classified as a variable miRNA in the PCA plot (labeled as “H” in the Fig. 3D).”

High inter-variability between individuals was observed for miR-19b-3p, miR-23a-3p, and miR-99a-5p.

“As expected, high inter-variability between individuals was observed for those miRNAs.”

The high variability seen in subject #5 was replicated well during the validation study.

“In particular, the high variability seen in subject #5 was replicated well during the validation study (Fig. 7C and D).”

The levels of most miRNAs in CSF were not variable during a 48-hour window in healthy individuals.

“In conclusion, we found that the levels of most miRNAs in CSF were not variable during a 48-hour window.”

• Also stated in: § Discussion · plain

  “Surprisingly, the vast majority of miRNAs were very stable at the 0-hour and 48-hour time points in our study.”

• Also stated in: § Discussion · boosted

  “In summary, we demonstrated that miRNAs in CSF are reliably detectable and very stable between 0- and 48-hour time points in most healthy individuals.”

There were no miRNAs whose expression level was variable across all 8 individuals.

“Interestingly, there were no miRNA whose expression level was variable across all 8 individuals.”

Among the miRNA biomarkers identified in the fibromyalgia study, miR-223-3p, miR-195-5p, and miR-125b-5p were found to be highly variable in our study.

“Among the miRNA biomarkers identified in this study, miR-223-3p, miR-195-5p, and miR-125b-5p were found to be highly variable in our study.”

miR-451a exhibited a 16-fold decrease only in subject #7 during the 48-hour study.

“In our study, miR-451a exhibited a 16-fold decrease only in subject #7 during our 48 hour study, while the level of miR-451a in other participants remained stable (Fig. 4H).”

miR-451a was identified as a variable miRNA in our PCA analysis.

“Although we identified miR-451a as a variable miRNA in our PCA analysis, this result was mainly caused by the intra-variability of a single participant.”

All 4 miRNAs (miR-30a-5p, miR-33a-5p, miR-139-5p, and miR-451a) showed the same up- or down-regulation expression pattern in subject #7 as reported by Wan et al.

“Interestingly, all 4 miRNAs showed the same up- or down-regulation expression pattern as reported by Wan et al.”

miR-19b-3p levels can be variable within the same individual even within a short 48-hour interval.

“However, our variability analysis indicated that miR-19b-3p levels can be variable within the same individual even within a short 48-hour interval (Fig. 5D).”

The variability of miR-451a in our PCA analysis was mainly caused by intra-variability of a single participant.

“Although we identified miR-451a as a variable miRNA in our PCA analysis, this result was mainly caused by the intra-variability of a single participant.”

The level of miR-451a in other participants remained stable.

“In our study, miR-451a exhibited a 16-fold decrease only in subject #7 during our 48 hour study, while the level of miR-451a in other participants remained stable (Fig. 4H).”

miRNAs in CSF are reliably detectable.

“In summary, we demonstrated that miRNAs in CSF are reliably detectable and very stable between 0- and 48-hour time points in most healthy individuals.”

The stability of most microRNAs in CSF suggests that microRNAs in CSF may be a good resource for identification of biomarkers.

“This suggests that microRNAs in CSF may be a good resource for the identification of biomarkers.”

The 12 microRNAs with variable levels may not be reliable biomarkers given their intrinsic variability even within the same individual.

“Our data strongly suggest that these microRNAs may not be reliable biomarkers given their intrinsic variability even within the same individual.”

miR-1246 and miR-374b-5p were empirically selected as the two endogenous reference genes based on the NormFinder algorithm.

“The two endogenous references, miR-1246 and miR-374b-5p, were empirically selected based on the NormFinder algorithm (Fig. 2).”

• cites [31] Andersen, C. L., Jensen, J. L. & Orntoft, T. F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64, 5245–5250, https://doi.org/10.1158/0008-5472.CAN-04-0496 (2004). sourceNot checked — no full text of the source

The variability of miRNA levels was not due to any experimental artifact or technical error.

“Therefore, these results demonstrate that the variability of miRNA levels was not due to any experimental artifact or our technical error.”

• AssumptionRests on an assumption: Any qRT-PCR assay with low signal intensity is likely to generate variable results.

miRNAs in CSF may be a reliable resource to find biomarkers for neurological diseases.

“This result suggests that miRNAs in CSF may be a reliable resource to find biomarkers for neurological diseases.”

• Also stated in: § Discussion · hedged

  “This finding suggests that miRNAs in CSF can be reliable biomarker candidates, because the constant expression level of a biomarker is critical for its high reliability and sensitivity.”

• Also stated in: § Discussion · hedged

  “This finding suggests that miRNAs in CSF can be reliable biomarker candidates for neurological diseases.”

• AssumptionRests on an assumption: The constant expression level of a biomarker is critical for its high reliability and sensitivity.

PCA plot is a useful tool to identify variable miRNAs.

“Taken together, our results demonstrate that PCA plot is a useful tool to identify the variable miRNAs”

PCA analysis alone cannot identify all variable miRNAs due to its inherent limitation.

“Although PCA clustering is an efficient method to determine the variability among miRNAs and individuals, PCA analysis alone cannot identify all variable miRNAs due to its inherent limitation.”

Subject #5 had a different miRNA expression pattern compared to other participants.

“Conversely, the distinct separation of the subject #5 from the group indicates that this individual had a different miRNA expression pattern compared to other participants.”

The SD of log2 RQV between individuals reflects high inter-individual variability.

“While a higher or lower RQV itself depicts the intra-individual variability during the 48-hour interval, the high SD of log2 RQV between individuals reflects the high inter-individual variability.”

Determining variable miRNAs using the SD of log2 RQV alone would fail to identify other variable miRNAs when participants' miRNA expression levels are all decreased or all increased.

“Determining the variable miRNAs using the SD of log2 RQV alone would fail to identify other variable miRNAs when participants’ miRNA expression levels are all decreased or all increased.”

CSF samples from young, healthy participants are likely to reflect a more physiological condition than postmortem CSF samples.

“Because we collected CSF from young, healthy participants, our CSF samples are likely to reflect a more physiological condition than postmortem CSF samples.”

The intrinsic variability in miRNA levels may explain the conflicting results from other biomarker studies.

“More importantly, our results suggest that the intrinsic variability in miRNA levels may explain the conflicting results from other biomarker studies.”

• AssumptionRests on an assumption: Inherent physiological characteristics, such as noise in miRNA gene expression over a short period, may be responsible for conflicting results among miRNA biomarker studies.

The CSF sample collection procedure was well designed to evaluate biological variability in miRNA levels.

“Therefore, our CSF sample collection procedure was well designed to evaluate a biological variability in miRNA levels.”

The selected 95 miRNA assays will cover most miRNAs reliably detectable in human antemortem CSF.

“Although we measured only 95 miRNAs, we believe that those selected 95 miRNA assays will cover most miRNAs reliably detectable in human antemortem CSF.”

The smaller number of miRNAs detected in CSF appears to be due to the use of antemortem CSF samples.

“This result appears to be due to the use of antemortem CSF samples in our experiments.”

High inter-individual variability in miRNA level could not be detected in PCA analysis if the level of a certain miRNA is increased in some participants while decreased to a similar extent in other participants.

“High inter-individual variability in miRNA level could not be detected in the PCA analysis if the level of a certain miRNA is increased in some participants while decreased to a similar extent in other participants.”

All participants stayed in the clinic during the CSF collection time period to minimize confounding environmental factors such as physical activities and diets.

“To minimize any possible confounding environmental factors, such as the participants’ physical activities and diets, we asked all participants to stay in the clinic during the CSF collection time period.”

• Also stated in: § Subjects information and CSF collection · plain

  “Subjects stayed in the clinic before and during the CSF collection.”

The combination of PCA plot and SD enables determination of intra- and inter-individual variability more effectively.

“Therefore, the combination of PCA plot and SD enables us to determine the intra- and inter-individual variability more effectively.”

Subject #5 was excluded from subsequent analyses because it was a clear outlier.

“Because the subject #5 was a clear outlier (Fig. 3A), we also excluded the data obtained from this subject in our subsequent analyses, unless otherwise stated.”

miR-195-5p, miR-125b-5p, miR-101-3p, and miR-19a-3p were identified as highly intra-variable miRNAs.

“Therefore, miR-195-5p, miR-125b-5p, miR-101-3p, and miR-19a-3p were identified as highly intra-variable miRNAs.”

The overall variability of a miRNA level will appear less in the PCA plot when the net effect of each alteration cancels out.

“In that case, the overall variability of this miRNA level will appear to be less in the PCA plot, because the net effect of each alteration may cancel out each other in the PCA analysis.”

Our large-scale study is the first to provide strong evidence that the basal levels of most miRNAs in CSF are not variable under normal physiological conditions.

“To our knowledge, our large-scale study is the first to provide strong evidence that the basal levels of most miRNAs in CSF are not variable under normal physiological conditions.”

• AssumptionRests on an assumption: The biological variability in miRNA levels has not been thoroughly investigated.
• AssumptionRests on an assumption: The intra-individual variability in CSF miRNA levels has not been addressed in previous studies.

The discrepancy between studies on miR-125b may be due to the intrinsic variability of miR-125b itself.

“Such data suggests that the discrepancy between studies may be due to the intrinsic variability of miR-125b itself.”