Diurnal Variations of Human Circulating Cell-Free Micro-RNA.
Author(s): Heegaard NH, Carlsen AL, Lilje B, Ng KL, Rønne ME, Jørgensen HL, Sennels H, Fahrenkrug J
Publication: PLoS One, 2016, Vol. 11, Page e0160577
PubMed ID: 27494182 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of microRNAs (miRNA, miR) in plasma that was collected every 3 h over a course of 24 h from 24 healthy volunteers.
Conclusion of Paper
Melatonin levels in plasma differed significantly among timepoints in the 24 h timecourse, with mean peak levels identified at 3:34 AM. A total of 79 miRNAs were reliably detected, of which 26 miRNAs were found to have a rhythmic expression (12 miRNAs were nocturnal and 14 miRNAs were diurnal). When normalized to miR-16 instead of the row average, nine miRNAs (2 nocturnal and 7 diurnal) displayed rhythmic expression. Many of the predicted targets of rhythmic miRNAs were established rhythmic genes, and genes involved in epigenetic regulation.
Studies
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Study Purpose
This study compared levels of miRNAs in plasma that was collected every 3 h over a course of 24 h from 24 healthy volunteers. Blood was collected from 24 healthy male volunteers into K3EDTA tubes every three hours over the course of a 24 h hospital stay. The stay included 15 h of light (08:00-23:00) and 9 h of dark (23:00-08:00); night sampling was performed under red light. Immediately after collection, plasma was separated by centrifugation at 1,500 g at 20°C for 10 minutes and frozen at -80°C. RNA was isolated from plasma using the Norgen Total RNA Purification Kit with the following modifications: addition of dTT cel-miR-54 and cel-miR-238 during the lysis step and addition of RNase inhibitor before elution. Isolated RNA was stored at -20°C. RNA was reverse transcribed with the TaqMan micro-RNA Reverse Transcription Kit and a mix of 48 different miR-specific stem-loop primers. cDNA was preamplified using TaqMan PreAmp kit and TaqMan MicroRNA Assays before quantification of two different panels of 48 miRNAs (96 total) by real-time PCR amplification in triplicate using 96 x 96 dynamic array chips. miRNAs with average quantification cycle (Cq) values >35 were excluded from analysis. miRNA levels were normalized to the average of the two spike-in miRNAs in the same specimen followed by row-specific Cq average or miR-16.
Summary of Findings:
Melatonin levels in plasma differed significantly among timepoints (P<0.001) in the 24 h timecourse, with mean peak levels identified at 3:34 AM. A total of 79 miRNAs were reliably detected (Cq ≤ 35 in >50% of specimens). Of these, 26 miRNAs were found to have rhythmic expression (P<0.05). Of these, 12 miRNAs exhibited peak levels during the night and 14 during the day. When normalized to miR-16 instead of the row average, nine miRNAs (2 nocturnal and 7 diurnal) displayed rhythmic expression (P<0.05). Many of the identified miRNAs have validated targets with rhythmic expression including SP1, MECP2, RASD1, CCND1, CEBPB, Per1, CDKN1A, IGF1R, ROCK2, and PTEN. The authors also found that many of the predicted targets of rhythmic miRNAs were genes involved in epigenetic regulation or intracellular MAPK signaling pathways. The authors identified 17 miRNAs that were predicted to target genes with validated rhythmic expression. Of these 17 miRNAs, only miR-24 (P=0.02) and miR-27 (P=0.02) were diurnal with the remaining 15 either not reliably detected (5 miRNAs) or not rhythmic in this study (10 miRNAs).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Data handling Normalized to Row Average
Normalized to miR-16
Biospecimen Acquisition Time of biospecimen collection Diurnal (08:00-23:00)
Nocturnal (23:00-08:00)