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miRNA Library Preparation Optimisation for Low-Concentration and Low-Volume Paediatric Plasma Samples.

Author(s): Rodgers O, Watson C, Waterfield T

Publication: Noncoding RNA, 2025, Vol. 11, Page

PubMed ID: 39997611 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study sought to optimize next-generation microRNA (miRNA) sequencing library preparation for pediatric plasma specimens by comparing library concentrations following extraction from 100 or 200 µL plasma using two different RNA extraction kits (miRNeasy Serum/Plasma Kit and the MagMAX miRVana Total Isolation Kit). Further, optimization of miRNA concentration, adapter and primer dilution and the number of PCR cycles was conducted using miRNA extracted with the miRNeasy Serum/Plasma Kit from 100 µL plasma. The effect of patient diagnosis, age and gender on the concentration of the miRNA library was also investigated using the optimized protocol. Blood was collected from infants (<90 days of age) with a fever (>38°C) admitted at two hospitals (FIDO Cohort) and from sick children (≤16 years of age) and healthy volunteers (≤15 years of age) (Rapid-19 Cohort); blood from all pediatric individuals were collected in EDTA tubes. Within 1 hour of collection, blood was centrifuged at 1500 g for 15 min to obtain plasma. Plasma was frozen at -80°C and transported to Queen’s University Belfast. RNA extraction kits were compared using six plasma pools that were created using plasma from infants (11 patients), healthy volunteers ≤15 years of age (3 patients) and sick children ≤16 years of age (3 patients); RNA was extracted from 100 µL and 200 µL aliquots using the miRNeasy Serum/Plasma Kit and the MagMAX mirVana Total RNA Isolation Kit. To increase miRNA concentration, 10 µL of extract was condensed with a Savant SPD1010 SpeedVac Concentrator and resuspended in 5 µL water prior to library preparation (Optimization 1). Libraries were constructed using the QIAseq miRNA Library with the 96 Index Kit and the QIAseq miRNA UDI Library Kit. miRNA libraries were analyzed for fragment size using a 5300 Fragment Analyzer System. Total RNA in RNA libraries was quantified using a Qubit 4 Fluorometer. Libraries were sequenced using a NextSeq 2000 instrument. Three optimization methods of QIAseq miRNA UDI Library production were evaluated using miRNA extracted with the miRNeasy Serum/Plasma Kit  and 100 µL of plasma from four patients. The tested library optimization methods were as follows: miRNA was condensed from 10 µL to 5 µL, 5’ and 3’ adaptors and RT primer were diluted to 1:10, 1:20, and 1:20, respectively, with 24 PCR amplification cycles  (Optimization 2); miRNA was condensed from 10 µL to 5 µL, 5’ and 3’ adaptors and RT primer were diluted to 1:5, 1:10, and 1:10, respectively, with 22 PCR amplification cycles (Optimization 3); and miRNA was condensed from 20 µL to 5 µL, 5’ and 3’ adaptors and RT primer were diluted 1:2.5, 1:5, and 1:5, respectively, with 22 PCR amplification cycles (Optimization 4).  The Optimization 2 method was then used to prepare sequencing libraries from 100 µL plasma from 92 pediatric patients.

   Summary of Findings:

   During the initial analysis, miRNA libraries had a very low concentration but were slightly higher when extraction was with the miRNeasy Serum/Plasma Kit (0-1.42 ng/µL, average 0.301 ng/µL) compared to the MagMAX mirVana Total RNA Isolation Kit (0-0.11 ng/µL, average 0.027 ng/µL). Library yields were higher when miRNA was extracted from 100 µL pooled plasma from sick children ≤16 years of age (miRNeasy 1.415 ng/μL and MagMAX 0.107 ng/μL), then from 200 µL pooled plasma from sick children (miRNeasy 0.054 ng/μL and MagMAX 0.002 ng/μL), or from 100 µL or 200 µL pooled plasma of healthy children (0.034 ng/μL and 0.303 ng/μL, respectively, with the miRNeasy Kit; 0.014 ng/μL and 0.0.16 ng/μL, respectively, with the MagMAX Kit) or pooled plasma of infants (0.00 ng/μL both kits, both volumes). Importantly, the fragment analyzer showed a peak at 50-60 bp, indicating a large amount of unbound adapters, and real-time PCR amplification of the spike-in UniSp6 control showed PCR inhibition, particularly in the specimen from infants. The average total reads and percentage unique reads were higher when RNA was extracted with the miRNeasy Kit rather than the MagMAX Kit (1.89x 10⁷ versus 5.76x 10⁶ total reads and 6.7% versus 2.7%) and when extraction with the miRNeasy Kit was from 100 µL rather than 200 µL (1.67x 10⁷ versus 7.95x 10⁶ total reads and 4.7% versus 4.6%), but the differences were not statistically significant. The authors selected an input of 100 µL of plasma and the miRNeasy Extraction Kits for optimization experiments using different combinations of extracted miRNA concentrations, adaptor and primer dilutions and numbers of PCR cycles. The Optimizations method evaluated, which diluted the adapters and primers (Optimization 2, 3 and 4), had less prominent unbound adapter peaks than were observed during the initial analysis (only miRNA concentration was performed) (Optimization 1). The miRNA library concentration was highest (8.19 ng/μL) when Optimization 4 was used (miRNA condensed from 20 µL to 5 µL, dilution of 5’ and 3’ adaptors and RT primer 1:2.5, 1:5, and 1:5, respectively, with 22 PCR amplification cycles), but this method required multiple miRNA extractions to obtain the required input. When comparing the three optimization methods that used miRNA condensed from 10 µL to 5 µL, Optimization 2 (dilution of 5’ and 3’ adaptors and RT primer 1:10, 1:20, and 1:20, respectively, with 24 PCR amplification cycles) yielded more concentrated miRNA library than Optimization 3 (dilution of 5’ and 3’ adaptors and RT primer 1:5, 1:10, and 1:10, respectively, with 22 PCR amplification cycles) or Optimization 1 (1.60 ng/μL versus 0.59 ng/μL and 0.30 ng/μL, respectively).  When the Optimization 2 method was tested with 92 pediatric patient specimens, PCR inhibition was low (CT value <18) in all but one specimen (CT =22), the average concentration was high (5.6 ng/μL), the failure (library with <0.5 ng/μL) rate was low (8.7%), and the spread of library concentrations was similar in specimens from patients diagnosed with viral, bacterial, and inflammatory diseases and healthy controls. The authors identified variation in miRNA library concentrations between the 8 different batches. miRNA library concentrations did not show clustering based on patient age (0-5, 5-10, and 10-16 years) or gender.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	0-5 years 5-10 years 10-16 years Infant (<90 days) ≤15 years of age ≤16 years of age
   Preaquisition	Patient gender	Female Male
   Preaquisition	Diagnosis/ patient condition	Infant with fever Sick child ≤16 years of age Healthy volunteers ≤15 years of age Bacterial infection Viral infection Inflammatory disease Healthy control
   Biospecimen Aliquots and Components	Aliquot size/volume	100 µL 200 µL
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy Serum/Plasma kit MagMAX miRVana Total Isolation kit
   Next generation sequencing Specific	Nucleic acid amplification	22 cycles 24 cycles
   Next generation sequencing Specific	Template/input amount	miRNA condensed from 20 µL to 5 µL miRNA condensed from 10 µL to 5 µL
   Next generation sequencing Specific	Reaction solution	Different primer and adapter dilutions compared

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