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MicroRNA in dried blood spots from patients with Aagenaes syndrome and evaluation of pre-analytical and analytical factors.

Author(s): Atneosen-Åsegg M, Melheim M, Almaas R

Publication: Pediatr Res, 2020, Vol. , Page

PubMed ID: 32932426 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the total miRNA yield and levels of individual miRNAs extracted from dried blood spots (DBS) between specimens obtained from one or three punches, punches from the core or periphery of the spot, punches from one or two drops of blood, and punches from cards freeze/thaw cycled one or ten times and identified the ratio of reagents during the extraction that resulted in the highest yields. The authors also compared normalization methods, normalized levels of three miRNAs in plasma and DBS specimens, and five miRNAs in newborn screening cards from patients with cholestasis-lymphedema syndrome/Aagenaes syndrome and healthy individuals. EDTA blood and capillary blood was obtained from eight healthy adults. Capillary blood was spotted onto a PerkinElmer226Newborn Screening Card, allowed to dry for 18 h at room temperature, and then stored in plastic bags at -20°C. Before use, cards were placed at room temperature for 30 min and 3.4 mm punches were obtained using a hole puncher. Plasma was obtained from EDTA blood by centrifugation at 300 x g for 10 min immediately after collection, aliquoted, and stored at -80°C. miRA was extracted from DBS punches and EDTA plasma using the miRNeasy Serum/Plasma Kit and stored at -80°C. miRNA was quantified using the Qubit microRNA Assay Kit and using custom TaqMan low density array (TLDA) real-time RT-PCR assays and normalized to miR-16. To test the effects of the QiAsol/chloroform ratio in the DBS extraction, the extraction was conducted using QiAsol/chloroform ratios of 300:100 and 500:200. To test the effects of punch location, punches were obtained from the center and periphery of the spot. To test the effects of blood volume, punches obtained from a single versus two drops of blood were compared. To investigate the stability of miRNA on dried blood spots during frozen storage, cards were freeze/thaw cycled once and ten times before RNA extraction. The authors also extracted miRNA from the newborn screening cards of four healthy newborns and five patients later diagnosed with Aagenaes syndrome that had been stored at -20°C for an average of 9.4±1.9 and 9.7±1.9 years, respectively.

   Summary of Findings:

   Significantly higher concentrations of miRNA were obtained from punches using a QiAsol/chloroform ratio of 500:300 than 200:100 (250±62 versus 181±42 ng/mL, P=0.033). As expected, extraction of miRNA from three punches rather than one punch resulted in a higher concentration of miRNA as assayed by fluorometry (890 ±222 ng/ml versus 250 ±87 ng/ml, P<0.0001) and lower raw CT values for all miRNA evaluated (P<0.0001, all), but had no effect on normalized miRNA levels. Location of the blood drop on the card (periphery versus center) and use of 1 versus 2 drops of blood had no impact on the concentration of miRNA obtained, CT values for miR-16, or normalized miRNA CT values. Freeze/thaw cycling of the punch cards ten times rather than once had no impact on the concentration of miRNA or on the normalized levels of any of the evaluated miRNAs. Levels of miR-140 and miR-210 were comparable in specimens extracted from DBS and plasma but miR-4429 levels were significantly higher in plasma than DBS (P<0.0001). Of the eight miRNAs considered as potential endogenous controls, miR-16 was identified as best using the Deta Cq, BestKeeper, and NormFinder algorithms and a combination of miR-16/miR-15b was considered best using the GeNorm algorithm. Of the 37 miRNAs identified in all newborn screening card punches, five were differentially expressed in patients later diagnosed with Aagenaes syndrome and controls.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • Other Preservative

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR
   RNA	Low density array
   RNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Newborn later diagnosed with Cholestasis-lymphedema syndrome/Aagenaes syndrome Newborn screening card from healthy child
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Biospecimen core Biospecimen periphery
   Biospecimen Aliquots and Components	Aliquot size/volume	1 drop of blood 2 drops of blood 1 punch 3 punches
   Biospecimen Aliquots and Components	Blood and blood products	Whole blood Plasma
   Storage	Freeze/thaw cycling	1 cycle 10 cycles
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy with QiAsol/chloroform ratio of 300:100 miRNeasy with QiAsol/chloroform ratio of 500:200
   Low density array Specific	Data handling	Normalized with Delta Cq Normalized with BestKeeper Normalized with NormFinder Normalized with GeNorm
   RT-PCR Specific	Targeted nucleic acid	96 miRNA panel

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