Serum microRNAs are promising novel biomarkers

Abstract

Background: Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers.

Methods and results: After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage.

Conclusions and significance: Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.

Figure 1. qRT-PCR can be used to monitor low microRNA levels specifically and sensitively.

A) Schematic representation of the qRT-PCR method. RNA is subjected to polyA polymerase reaction. Then, a universal RT reaction is performed that allows the amplification of all microRNAs as well as mRNAs. The PCR amplification is performed using a reverse primer complementary to part of the oligodT primer and a forward primer, which is homologous to a stretch in the microRNA sequence; in addition, the amplification reaction contains a TaqMan probe that covers part of the oligodT primer sequence and some nucleotides complementary to the 3′ sequence of the microRNA. B) Each synthetic RNA (hsa-let 7a, c & d) was subjected to three independent qRT-PCR amplifications, where in each reaction there were present primers specific to only one of the three family members. PCR amplification was observed only in the reaction where the primer matches the synthetic RNA. RNA amounts are described as percentages, each relative to the level observed in the reaction containing primers matching the synthetic microRNA. ND is non-detectable. C) All three synthetic microRNAs were mixed and subjected to qRT-PCR in the presence of the let-7d primer-probe set. In parallel, the same amount of synthetic let-7d as used in the mixture was subjected alone to qRT-PCR. These parallel PCR reactions were repeated using reducing concentrations of let-7d synthetic microRNA. At all tested concentrations of let-7d, it was amplified equivalently whether alone or in the presence of homologous family members. The C_T of let-7d is proportional to the input microRNA amount. D) Reducing concentrations (100-0.03%) of total RNA extracted from liver tissue (Ambion Inc., No. 0360093B#) were mixed with total RNA extracted from brain tissue (Ambion Inc., No. 016P040305030A#) and the mixtures subjected to qRT-PCR, where each reaction contained a primer specific to hsa-miR-122a. Hsa-miR-122a was detected linearly, even in samples where only 0.03% liver RNA was spiked into brain RNA. When no liver RNA was introduced (100% brain RNA), hsa-miR-122a was not detected. E) The amounts of 32 different microRNAs in an RNA sample were examined on two independent occasions (by two different researchers) using the qRT-PCR platform (microRNA level is represented as C_T value). The two profiles were within less than 1 C_T difference of one another.

Figure 2. MicroRNAs are present in bodily fluids.

A) microRNA levels in serum samples taken from 2 healthy individuals were measured. The levels of 18 different microRNAs (blue circles, cycle thresholds (C_T) values) and the 4 synthetic RNA ‘spike-ins’ (in the lower left part of the graph) were found to be similar. B) To demonstrate that our extraction and evaluation methods can be applied to other body fluids, the same set of 20 microRNAs examined in serum were assessed in urine samples from 2 healthy individuals. Some microRNAs were undetectable in the urine samples and therefore are not shown. Notably, urine and serum samples demonstrate different microRNA abundance profiles.

Figure 3. MicroRNAs are stable during serum handling.

A) microRNA stability in serum samples was monitored by extracting RNA from serum samples kept for 1, 2 or 4 h at room temperature before freezing. The levels of 20 different microRNAs (blue circles), as well as of the 4 synthetic RNA ‘spike-ins’ (in the lower left part of the graph), were found to be similar across the 4 h time period. B) microRNA stability in serum samples was monitored by extracting RNA from serum samples before and after freezing. The levels of 20 different microRNAs (blue circles), as well as of the 4 synthetic RNA ‘spike-ins’ (in the lower left part of the graph), were found to be similar following re-freezing and re-thawing of the sample.

Figure 4. Differential amounts of four microRNAs in the sera of pregnant vs. non pregnant women.

Box plots comparing microRNA levels in the sera of 10 non pregnant women (A), 10 women in the first trimester (B), and 10 women in the third trimester (C). microRNA level is specified as 50-C_T, where C_T is the cycle threshold of the PCR reaction. Results were normalized by subtracting the global microRNA level in the sample (average C_T of the 6 microRNAs chosen for normalization) from the level (C_T) of each microRNA. A) The three placental microRNAs (miR-527, miR-520d-5p and miR-526a) are highly abundant in the sera of pregnant women and their levels rise as pregnancy progresses. Hsa-let-7d levels are also shown; this was one of the 6 microRNAs chosen for normalization as this microRNA exhibits similar abundance across the three groups. B) microRNA miR-141 and miR-149 levels are mildly upregulated during pregnancy.

Figure 5. “Pregnancy classification” according to the levels of three microRNAs in the sera of pregnant vs. non pregnant women.

Discrimination of pregnant women from non pregnant women based on microRNA levels in their sera. Blue circles represent non pregnant women and red triangles represent pregnant women. The location of each symbol in the plot represents the collective expression of all three microRNAs in a given serum. The y axis indicates the amount of mir-527, and the x axis indicates the average level of miR-520d-5p and miR-526a.