AbstractThe profiling of circulating nucleic acids is a key step toward the development of noninvasive, blood-based molecular diagnostic tests. The discovery that serum and plasma contain a large amount of stable miRNAs derived from various tissues/organs has lead to multiple studies on circulating miRNA expression1,2. However, the clinical effectiveness of circulating microRNAs as biomarkers is likely to be affected by a range of pre-analytical variables such as RNA extraction efficiency and methodological issues involved in platform-specific sample preparation for miRNA profiling. Reproducible isolation of cell-free miRNAs is a technical challenge for a number of reasons. First, plasma and serum are biospecimens that have a very high concentration of protein that could potentially interfere with sample preparation and the detection assay. Secondly, the yield of RNA from small volume plasma or serum samples (< 1 mL) usually falls below the limit of accurate quantification by spectrometry and calls for an alternative way to assess the efficiency of RNA recovery. In order to develop an optimized protocol, we compared the performance of three different extraction protocols and included several synthetic miRNA spike-ins for sample quality and extraction efficiency assessment. Further, we compared the ability to profile the miRNAs from these samples using both microarray and LNA-based qRT-PCR platforms. Our data shows that the protocol used for miRNA extraction can interact with the detection platform. We were able to develop an optimized protocol that shows good alignment between microarray data and RT-PCR assays (miRCURY LNA™ RT-PCR assays).

MethodsRNA IsolationHuman blood serum samples from healthy volunteers were pooled before extraction to reduce variability and divided into 250-µL aliquots.

Three isolation methods were employed:

1) Extraction with Trizol/chloroform followed by isopropanol precipitation

2) Extraction with Trizol/chloroform followed by isopropanol precipitation and additional purification on Qiagen RNeasy columns

3) RNA isolation using the QIAamp Circulating Nucleic Acid kit (miRNA protocol)

For each isolation method we used 500 ng of yeast tRNA as a carrier and a mixture of nonhuman, single-stranded synthetic miRNAs (C. elegans cel-miR-39 and cel-miR-54, 20 fmols each). Final elution volume for all the isolation methods was 85 µL.

RT-PCRReverse transcription was performed using the Exiqon Universal cDNA Synthesis kit and 5 µL of each sample. PCR reactions were performed in individually using Exiqon SYBR® Green Master mix and custom miRNA assays according to the vendor’s protocol. All PCR reactions were run on an ABI7900 instrument without ROX dye.

RNA Labeling, Hybridization on GeneChip® Microarray and Data AnalysisSamples (~80 µL) were dried down, reconstituted in 8 µL water and labeled using Genisphere® Flashtag™ HSR kit according to the vendor’s procedure. Hybridization on Affymetrix GeneChip® miRNA arrays (v 1.0) was performed for 42 h; microarray wash/scan was performed as described in Genisphere protocol. Data analysis was done using the miRNA QC tool (Affymetrix).

Results and DiscussionRNA IsolationBoth plasma and serum are widely used specimen types for circulating miRNA analysis; however, there is considerable sample-to-sample variability in both protein and lipid content of plasma and serum samples. This could affect the efficiency of RNA extraction and introduce inhibitors of downstream assays. Therefore, the choice of an efficient RNA isolation method compatible with downstream miRNA assay is of foremost importance.

After studying several related publications, we decided to test three isolation methods. The first method—extraction with Trizol/chloroform followed by isopropanol precipitation—was demonstrated to preserve the miRNA fraction of total RNA most efficiently. Doing the same extraction with an additional purification using RNeasy columns, we hoped to further remove any proteins and lipids that may contaminate RNA in serum samples. We have also tested the commercially available QIAamp protocol that is specifically designed for circulating nucleic acids (including miRNAs) and features a separate Proteinase K treatment. The extractions were done in triplicate (quadruplicate for the QIAamp protocol).

UV measurements taken after the extraction shows low concentration of nucleic acid material with questionable purity (Table1).

To evaluate the sample quality and extraction efficiency, we used RT-PCR (Exiqon miRNA assays). Custom assays for spike-ins cel-miR-39 and cel-miR-54 were employed to estimate the extraction success. Catalog assays for two human microRNAs hsa-miR-16 and hsa-miR-223 were utilized to estimate the resulting sample quality as both miRNAs are expressed at high levels in plasma and serum.3

As can be seen from Figure 1, the highest extraction efficiency (lowest Ct values for the spike-ins cel-miR-39 and cel-miR-54) was observed for Trizol/precipitation protocol. Additional column purification led to ~4 times lower amount of detected microRNA spike-ins; and the QIAamp protocol appeared to be ~50 times less successful in extraction than Trizol/precipitation procedure.

It is interesting to note however, that while the naturally occurring human microRNAs hsa-miR-16 and hsa-miR-223 follow the same trend in Ct values, the Ct difference between these microRNAs and C. elegans spike-ins becomes smaller with the increase in extraction protocol “complexity.”

Optimization of miRNA Extraction from SerumZinaida Sergueeva, Sally Dow, Heather Collins and Mark L. ParrishCovance Genomics Laboratory, Seattle, Washington

RNA Labeling, Hybridization and Data AnalysisAll samples were labeled and hybridized on Affymetrix GeneChip® miRNA v 1.0 arrays according to the Genisphere protocol. The protocol employs five QC spike-in controls to monitor the labeling and hybridization process on the array. As can be seen from Table 2 and Figure 2, the raw intensities for the array QC spike-in controls 23, 29 and 31 are significantly lower for the Trizol/precipitation protocol while for two other protocols all array QC spike-in controls perform in a similar way.

We have also looked at the corresponding probe signal intensities for the extraction spike-ins cel-miR-39 and cel-miR-54 and two highly expressed human microRNAs – hsa-miR-16 and hsa-miR-223. Table 3 shows that microarray data for the extraction spike-ins cel-miR-39 and cel-miR-54 are concordant with qPCR analysis: signal intensities are higher for the Trizol/precipitation protocol and noticeably lower for the QIAamp protocol. However, for the naturally occurring miRNAs the opposite trend is true: signal intensities are higher and less variable for the two protocols that involve additional column purification.

Reproducibility between technical replicates is better for samples isolated with Trizol/RNeasy cleanup and QIAamp protocols (Figure 3).

Furthermore, we compared the detection level among the samples extracted with all three protocols. As can be seen from Figure 4, additional purification leads to detecting a higher number of miRNAs. Statistical analysis shows that this difference is very significant (data not shown).

ConclusionThe experimental data collected during this study confirm that Trizol/precipitation protocol is not suitable for the microarray sample preparation from blood serum; it retains some inhibitors that affect the labeling and hybridization process. Both extraction protocols that incorporate column-based purification—Trizol/RNeasy cleanup and QIAamp—provide good quality microarray data on both microarray and qPCR platforms.

References1. X. Chen, Y. Ba, L. Ma, X. Cai, Y. Yin, K. Wang, J. Guo, Y. Zhang, J. Chen, X. Guo, Q. Li, X. Li, W. Wang, Y. Zhang, J. Wang, X. Jiang, Y. Xiang, C. Xu, P. Zheng, J. Zhang,

R. Li, H. Zhang, X. Shang, T. Gong, G. Ning, J. Wang, K. Zen, J. Zhang, C.Y. Zhang. Characterization of microRNAs in serum : a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 10 (2008) 997-1006.

2. P.S. Mitchell, R.K. Parkin, E.M. Kroh, B.R. Fritz, S.K. Wyman, E.L. Pogosova-Agadjanyan, A. Peterson, J. Noteboom, K.C. O’Briant, A. Allen, D.W. Lin, N. Urban, C.W. Drescher, B.S. Knudsen, D.L. Stirewalt, R. Gentleman, R.L. Vessela, P.S. Nelson, D.B. Martin, M. Tewari. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl. Acad. Sci. USA 105 (2008) 10513-10518.

3. E.M. Kroh, R.K. Parin, P.S. Mitchell, M. Tewari. Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods 50 (2010) 298-301.

Table 1. Nanodrop UV Measurements for Serum Samples after the Extraction

Figure 1. RT-PCR data for serum samples extracted with three different protocols and analyzed using microRNA Exiqon assays for cel-miR-39 (red dots), cel-miR-54 (blue dots), hsa-miR-16 (yellow dots) and hsa-miR-223 (black dots).

Table 2. Signal Intensities for Array QC Spike-In Controls

Figure 2. Signal intensities for array QC spike-in controls.

Table 3. Signal Intensities for microRNAs

Figure 4. Number of miRNAs detected in serum samples extracted with all three protocols.

Figure 3. Signal intensity correlation between technical replicates for the serum samples isolated with Trizol/precipitation (A), Trizol/RNeasy cleanup (B) and QIAamp (C) protocols.

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