Dataset Information

Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.

ABSTRACT:

Background

The measurement of circulating miRNAs has proven to be a powerful biomarker tool for several disease processes. Current protocols for the detection of miRNAs usually involve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficient input material.

Objective

Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction.

Methods

Three ?l of serum or plasma was subjected to three cycles of high and low temperatures (heat/freeze cycles) followed by miRNA arrays.

Results

Our results indicate that miRNA detection following this process is highly reproducible when comparing multiple samples from the same subject. Moreover, this protocol increases the reproducibility of miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles. Importantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highly comparable, indicating that this heat/freeze process effectively eliminates differences in detection between serum and plasma samples that have been reported using other sample preparation methodologies.

Conclusion

We propose that this method is a potent alternative to current RNA extraction protocols, substantially reducing the amount of sample necessary for miRNA detection while simultaneously improving miRNA detection and reproducibility.

SUBMITTER: Mariner PD

PROVIDER: S-EPMC6198569 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.

Mariner Peter D PD Korst Armin A Karimpour-Fard Anis A Stauffer Brian L BL Miyamoto Shelley D SD Sucharov Carmen C CC

MicroRNA (Shariqah, United Arab Emirates) 20180101 2

<h4>Background</h4>The measurement of circulating miRNAs has proven to be a powerful biomarker tool for several disease processes. Current protocols for the detection of miRNAs usually involve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficient input material.<h4>Objective</h4>Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction.<h4>Meth ...[more]

PMID: 29658445

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Project description:AimThe study was designed to assess the possibility of using circulating miRNAs (serum miRNAs) as diagnostic biomarkers in colorectal cancer (CRC) and to identify their possibility as candidates for targeted therapy.MethodsThe study involved two sample sets: 1- a training set which included 90 patients with colorectal related disease (30 with CRC, 18 with inflammatory bowel disease (IBD), 18 with colonic polyps (CP) and 24 with different colonic symptoms but without any colonoscopic abnormality who were enrolled as control group) and 2- a validation set which included 100 CRC patients. Serum miRNAs were extracted from all subjects to assess the expression profiles for the following miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-146a, miR-223, miR-24, miR-454, miR-183, miR-135a, miR- 135b and miR- 92a) using the custom miScript miRNA PCR-based sybergreen array. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the studied miRNAs for colorectal cancer diagnosis.ResultsData analysis of miRNA from the training set showed that; compared to control group, only miR-19b was significantly up-regulated in patients with IBD group (fold change = 5.24, p = 0.016), whereas in patients with colonic polyps, miR-18a was significantly up-regulated (fold change = 3.49, p-value = 0.018). On the other hand, miR-17, miR-19a, miR-20a and miR-223 were significantly up-regulated (fold change = 2.35, 3.07, 2.38 and 10.35; respectively and p-value = 0.02, 0.015, 0.017 and 0.016; respectively in CRC patients. However, the validation set showed that only miR-223 was significantly up-regulated in CRC patients (fold change = 4.06, p-value = 0.04).ConclusionAberrant miRNA expressions are highly involved in the cascade of colorectal carcinogenesis. We have found that (miR-17, miR-19a, miR-20a and miR-223) could be used as diagnostic biomarkers for CRC. On the other hand, miR-19b and miR-18a could be used as diagnostic biomarkers for CP and IBD respectively.

Project description:Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample's life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4°C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freeze-thaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon's miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples.

Dataset Information

Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.

Background

Objective

Methods

Results

Conclusion

Publications

Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.

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