microRNA Biomarker Discovery and High-Throughput DNA Sequencing Are Possible Using Long-term Archived Serum Samples

Rounge, Trine B.; Lauritzen, Marianne; Langseth, Hilde; Enerly, Espen; Lyle, Robert; Gislefoss, Randi Elin

doi:10.1158/1055-9965.epi-15-0289

Cited by 38 publications

(47 citation statements)

References 43 publications

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“…While the long-time storage did not reduce miRNA yields the preanalytical conditions like different clotting times had a significant effect on miRNA abundances. 27 Further evidence for the stability of miRNAs in stored samples came from a real-time PCR analysis that was performed on a panel of 8 miRNAs from samples that were stored for 12 months at ¡80 C. 28 Independent of the influence of the duration of the storage it is important to address the difference between various collection protocols. Although the majority of the protocols is on collections of sera, there is a multitude of variations including scenarios that required the collection of plasma or of whole blood, which is later used for serum or plasma isolation, scenarios that largely vary in terms of storage duration and storage temperature of either blood, serum or plasma, and protocols that vary in terms of the methods used for RNA isolation.…”

Section: Discussionmentioning

confidence: 99%

Sources to variability in circulating human miRNA signatures

et al. 2017

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An increasing number of studies propose circulating microRNAs (miRNAs) as biomarkers for a large number of human diseases including cancer, cardiovascular diseases, neurologic pathologies and others. To further validate miRNA as biomarkers it is indispensable to understand the variability of circulating miRNAs in healthy individuals. We determined the longitudinal miRNomes of 90 serum samples from the Janus Serum Bank in Norway, which have been stored between 23 and 40 y at -25 °Celsius. We profiled 3 serum samples with microarrays for 30 individuals, each. For each individual the samples were collected with a time interval of approximately 5 y. This design allowed insights into inter-individual variability, age dependent miRNA variability and the impact of storage length and pre-processing. A significant proportion of the miRNome was affected by the age of the blood donor and a not negligible, albeit small, part of the miRNome by the storage time. A substantial part of miRNAs was differentially abundant between individuals, independent of the time when samples were collected. Stepwise filtering of the 529 miRNAs that were detected in the serum samples showed 168 miRNAs with differential abundance depending on the time point analyzed, 56 miRNAs differentially abundant between individuals, and 169 miRNAs with an abundance depending on the sampling procedure. While these groups of miRNAs contain generally interesting and biologically important miRNAs, the remaining 135 miRNAs constitute very promising biomarker candidates as they show an overall low variability between healthy individuals, a likewise overall low variability across a longer life span, and a high independence of the sampling process and the storage length.

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Section: Discussionmentioning

confidence: 99%

Sources to variability in circulating human miRNA signatures

et al. 2017

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“…Under these conditions all unstable RNAs have been degraded. We have shown that the total amount of miRNA was affected by the processing of the serum and to a lesser extent by storage time 21 . Also, the number of other sncRNAs decrease with storage 47 .…”

Section: Discussionmentioning

confidence: 91%

Circulating small non-coding RNAs associated with age, sex, smoking, body mass and physical activity

Rounge

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Keller

et al. 2018

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Non-coding RNAs (ncRNA) are regulators of cell functions and circulating ncRNAs from the majority of RNA classes, such as miRNA, tRNA, piRNAs, lncRNA, snoRNA, snRNA and miscRNAs, are potential non-invasive biomarkers. Understanding how non-disease traits influence ncRNA expression is essential for assessing their biomarker potential.We studied associations of common traits (sex, age, smoking, body mass, physical activity, and technical factors such as sample storage and processing) with serum ncRNAs. We used RNAseq data from 526 donors from the Janus Serum Bank and traits from health examination surveys. We identified associations between all RNA classes and traits. Ageing showed the strongest association with ncRNA expression, both in terms of statistical significance and number of RNAs, regardless of RNA class. Serum processing modifications and storage times significantly altered expression levels of a number of ncRNAs. Interestingly, smoking cessation generally restored RNA expression to non-smoking levels, although for some isomiRs, mRNA fragments and tRNAs smoking-related expression levels persisted.Our results show that common traits influence circulating ncRNA expression. Therefore it is clear that ncRNA biomarker analyses should be adjusted for age and sex. In addition, for specific ncRNAs identified in our study, analyses should also be adjusted for body mass, smoking, physical activity and serum processing and storage.

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“…f The total DNA input in samples that failed and passed 94% PCT-QCCR. g, h, i The relationship between total DNA input and call rates for Axiom, standard Illumina and Illumina FFPE protocols, respectively sequencing [9], genotyping using real-time PCR [3] and SNP array (Affymetrix) analyses [10] of Janus Serum Bank samples have been successful. However, another study concluded that genotyping with DNA extracted from serum did not provide reliable data using highthroughput multiplex approaches, but was successful using Taqman [11].…”

Section: Discussionmentioning

confidence: 99%

Ultralow amounts of DNA from long-term archived serum samples produce quality genotypes

et al. 2019

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While genotyping studies are scavenging for suitable samples to analyze, large serum collections are currently left unused as they are assumed to provide insufficient amounts of DNA for array-based genotyping. Long-term stored serum is considered to be difficult to genotype since preanalytical treatments and storage effects on DNA yields are not well understood. Successful genotyping of such samples has the potential to activate large biobanks for future genome-wide association studies (GWAS). We aimed to evaluate genotyping of ultralow amounts of DNA from samples stored up to 45 years in the Janus Serum Bank with two commercially available platforms. 64 samples, with various preanalytical treatments, were genotyped on the Axiom Array from Thermo Fisher Scientific and a subset of 24 samples with slightly higher yield were genotyped on the HumanCoreExome array from Illumina. Our results showed that about 80% of the serum samples produced call rates with the Axiom arrays that would be satisfactory in GWAS. The mean DNA yield was 5.8 ng as measured with PicoGreen, 3-6% of recommended yield. The failed samples had on average lower input amounts of DNA. All serum samples genotyped on the HumanCoreExome with a standard and FFPE protocol produced GWAS satisfactory call rates, with mean 97.57% and 98.35% call rates, respectively. The mean yield was 10.65 ng, 6% of the recommendations. Successful array-based genotyping of ultralow DNA yields from serum samples stored up to 45 years is possible. These results demonstrate the potential to activate large serum biobank collections for future studies.

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microRNA Biomarker Discovery and High-Throughput DNA Sequencing Are Possible Using Long-term Archived Serum Samples

Cited by 38 publications

References 43 publications

Sources to variability in circulating human miRNA signatures

Sources to variability in circulating human miRNA signatures

Circulating small non-coding RNAs associated with age, sex, smoking, body mass and physical activity

Ultralow amounts of DNA from long-term archived serum samples produce quality genotypes

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