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Preanalytical considerations in quantifying circulating miRNAs that predict end-stage kidney disease in diabetes.

Author(s): Satake E, Krolewski B, Kobayashi H, Md Dom ZI, Ricca J, Wilson JM, Hoon DS, Duffin KL, Pezzolesi MG, Krolewski AS

Publication: JCI Insight, 2024, Vol. 9, Page

PubMed ID: 38912578 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the miRNA profile obtained by NGS of whole plasma using the HTG EdgeSeq platform with the profile obtained by sequencing extracted RNA using the HTG EdgeSeq, SeqMatic, or LC Sciences platforms or by real-time RT-PCR. This study used plasma and previously extracted RNA collected from eight patients (7 men and 1 woman) with type I diabetes and extracted RNA from plasma pools that included specimens from 40 type 1 diabetes patients.  RNA extraction was performed using the miRNeasy Serum/Plasma Kit. Levels of 2083 miRNAs were quantified in matched plasma and extracted RNA specimens from eight patients using the HTG EdgeSeq miRNA sequence platform. miRNAs were also quantified by sequencing the extracted RNA from those same eight patients by SeqMatics using SeqMatic’s TailorMix miRNA Sample Preparation Kit V2 Kit and a HiSeq 2000 DNA sequencer and by LC Sciences using the TruSeq Small RNA Sample Prep Kit and a HiSeq 2500 sequencer.  Levels of 1805 miRNAs were quantified using real-time RT-PCR and the miRNome miScript miRNA PCR Array and the RNA extracted from the plasma pools. 

   Summary of Findings:

   Using the HTG EdgeSeq platform, 2002 of 2083 miRNAs were detected directly in whole plasma; however, only 46% (930) of those miRNAs were detected when isolated RNA was sequenced using the HTG EdgeSeq platform, with 53 miRNAs (5.7%) expressed at lower levels and 46 (4.9%) expressed at higher levels in extracted RNA compared to levels in whole plasma. Of the 2002 miRNAs detected by HTG EdgeSeq in whole plasma, only 13% (262) and 22% (439) were detected in extracted RNA that was sequenced using the SeqMatic and LC Sciences RNA-Seq platforms, respectively, with detected miRNAs found at both higher and lower levels in isolated RNA compared to whole plasma. A total of 1442 miRNAs found in whole plasma were included in the real-time RT-PCR assay, of which 379 (27%) were detected in RNA extracted from plasma, but once again, expression levels differed between the sample types/platforms.  All eight high-risk ESKD miRNAs were detected in extracted RNA using the HTG EdgeSeq platform, but levels were significantly lower and not correlated with those in plasma. Only two of the eight high-risk ESKD miRNAs were detected in extracted RNA using either the LC Sciences RNA Seq or the real-time RT-PCR assay, and only one of the eight was detected in extracted RNA using the SeqMatic RNA Seq assay. All nine protective ESKD miRNAs were detected in extracted RNA by HTG EdgeSeq, seven were detected using either the LC Sciences RNA Seq or the real-time RT-PCR, and six were detected using the SeqMatic RNA Seq assay. Importantly, levels of some miRNAs were correlated between plasma and extracted RNA, but the authors state the strength of the correlations was highly variable.  

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Diabetes Type 1

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	RNA extraction performed No RNA extraction performed
   Next generation sequencing Specific	Technology platform	HTG EdgeSeq platform SeqMatic RNA seq LC Sciences RNA Seq Real-time RT-PCR

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2. Study Purpose

   This study investigated if plasma miRNA concentrations are correlated (i) with plasma storage duration, (ii) between plasma specimens thawed once versus three times, (iii) between collection timepoints in patients who did or did not progress to ESKD, or (iv) with patient age, BMI, or HbA1c levels. This study previously collected plasma from patients with type I (n=96) or II (n=140) diabetes (further collection details not provided). Levels of 2083 miRNAs were quantified directly in plasma (no RNA extraction) using the HTG EdgeSeq miRNA sequence platform. miRNA levels underwent quantile normalization with sample weights using the edgeR and limma R packages from Bioconductor. The threshold for miRNA detectability was >1 count per million in > 90% of the specimens. To assess the stability of miRNAs following storage, miRNAs were profiled in the plasma of 145 type II diabetes patients that had been stored for 4-14 years at -80°C both after a single and three freeze-thaw cycles.  The effects of patients BMI, age, gender and HbA1c levels on miRNA profile were also assessed using the specimens from the 145 patients with type II diabetes.  To better understand the effect of ESKD progression on the concentrations of miRNAs, the miRNA profile was compared between plasma collected at an initial visit and a follow-up visit (7-15 years later) from 96 type I diabetes patients of whom 44 progressed to ESKD and 52 did not. 

   Summary of Findings:

   While significant correlations were observed between levels of some miRNAs and the duration of -80°C storage, the majority of miRNAs were not significantly correlated with plasma storage duration.  Weak (-0.3<r<0.3), but not significant, correlations were observed between the levels of three (of the eight) high-risk ESKD-associated miRNAs and plasma storage duration.  Correlations between levels of the nine ESKD protective miRNAs and plasma storage were even weaker than the high-risk ESKD-associated miRNAs.  Levels of miRNAs were strongly correlated between specimens freeze-thaw cycled once and three times (r=0.882).

   Levels of the ESKD-associated miRNAs were not significantly different between the baseline and follow-up visits in plasma from the 52 patients with type I diabetes who did not progress to ESKD, but significant increases in five of eight ESKD-associated miRNAs and decreases in four of nine ESKD protective miRNAs were observed among the 44 patients who did progress to ESKD, but no other miRNAs were significantly increased.  The concentrations of the 2,002 miRNAs and the 17 ESKD-associated miRNAs in whole plasma were not associated with patient age, BMI, or HbA1c nor did they differ between specimens from men and women. 

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Diabetes Type 2
   • Diabetes Type 1

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Preaquisition	Patient age	60 ± 5 years
   Preaquisition	Patient body mass index	BMI range not specified
   Preaquisition	Prognostic factor	HbA1c 7.7 ± 1.7%
   Biospecimen Acquisition	Time of biospecimen collection	Initial visit Follow-up visit (7-15 years later)
   Preaquisition	Diagnosis/ patient condition	Type II diabetes with ESKD progression Type II diabetes without ESKD progression
   Storage	Storage duration	4-14 years
   Storage	Freeze/thaw cycling	1 cycle 3 cycles

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