Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina’s TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.     

Determination of plasma microRNA for early detection of gastric cancer

Gastric cancer is the fourth most prevalent malignancy worldwide and remains the second most common cause of cancer-related death globally. Understanding the molecular structure of gastric carcinogenesis might identify new diagnostic and therapeutic strategies for this disease. Thus, early detection of gastric cancer is a key measure to reduce the mortality and improve the prognosis of gastric cancer. There have recently been several reports that microRNAs (miRNAs) circulate in highly stable, cell-free forms in blood. Because serum and plasma miRNAs are relatively easy to access, circulating miRNAs also have great potential to serve as non-invasive biomarkers. Although a number of miRNAs associated with gastric cancer have been identified, the underlying mechanism of these miRNAs in tumorigenesis and tumor progression remains to be investigated. The purpose of this study is to identify the potential of serum miRNAs as biomarkers for early detection of gastric cancer patients. RNA was isolated using the High Pure miRNA Isolation Kit (Roche) following the manufacturer’s protocol. cDNA and preamplification protocols were obtained from the isolated plasma miRNAs. The BioMark? 96.96 Dynamic Array (Fluidigm Corporation) for real-time qPCR was used to simultaneously quantite the expression of 740 miRNAs. All statistical analyses were performed using the Biogazelle’s qbase PLUS 2.0 software. In this study, among 740 miRNAs that we analyzed only miR-195-5p was significantly (p < 0.05, fold changes = 13, 3) down-regulated in gastric cancer patients compared with control. We demonstrated that miR-195-5p is a novel tumor suppressor miRNA and may contribute to gastric carcinogenesis. The miRNA expression profile described in this study should contribute to future studies on the role of miRNAs in gastric cancer.     

Influence of Plasma Processing on Recovery and Analysis of Circulating Nucleic Acids

Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp^® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.     

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.     

Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles

Background

Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA.

Methods

3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed.

Results

RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed.

Conclusion

We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.     

Real-time quantification of microRNAs by stem-loop RT-PCR

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.     

Purification of high-quality micro RNA from the heart tissue

Micro RNAs (miRNA) are an abundant class of small RNAs that regulate the stability and translation of cognate mRNAs. MiRNAs are potential diagnostic markers, moreover, they play an essential role in the development of various heart disesases. In case of limited tissue material, such as, e.g. human biopsies, purification of miRNAs with sufficient yield is critical. Reproducible expression analysis of miRNAs is highly dependent on the quality of the RNA, which is often difficult to achieve from fibrous tissue such as the heart. Several companies developed general purification kits for miRNAs, however, none of them are specialized to fibrotic tissues. Here we describe an optimized miRNA purification protocol that results in high miRNA yield as compared to other methods including trizol-based and column-based protocols. By using our improved protocol, miRNA obtained from heart tissue gave more reproducible results in QRT-PCR analysis and obtained more significant calls (172 vs. 118) during DNA microarray analysis when compared to the commercially available kit. In addition to the heart tissue, the present protocol can be applied to other fibrotic tissues, such as lung or skeletal muscle to isolate high-purity miRNA.     

Real-time quantification of microRNAs by stem–loop RT–PCR

A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.     

Impact of preovulatory estradiol concentrations on conceptus development and uterine gene expression

The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus-oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16-27 bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P<0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes.     

Using a commercial DNA extraction kit to obtain RNA for RT-PCR from starchy rice endosperm

The extraction of RNA from a starchy plant material, such as many common food grains, is difficult, and especially so from the mature endosperm of rice. Most commercial RNA kits are not suitable for starchy materials. Traditional RNA extraction procedures, in addition to being laborious and time consuming, leave hazardous organic wastes that result in expensive disposal costs. Interestingly, the numerous commercial DNA isolation kits now available often include directions for eliminating co-isolated RNA. This indicated an approach to obtain the generally unwanted RNA by-product by treating the total extraction product to intentionally retain RNA. A method was developed by which a two-step DNase procedure was applied to the product of the Cartagen Food DNA extraction kit that eliminated the DNA but left the co-extracted RNA. This modified procedure was compared with several other commercial and standard methods that are promoted as being able to work under high polysaccharide conditions. Successful extraction was determined by the production and amplification of cDNA by RT-PCR of actin. Extraction was successful from milled rice, as well as from cornmeal and wheat flour. The modification provides an RNA extraction method that is quick, easy, and inexpensive, and also eliminates the production of hazardous wastes.     

Instability of miRNA and cDNAs derivatives in RNA preparations

Micro RNAs (miRNAs) are small RNA molecules, which function as important regulators of gene expression. We found that RNA preparation methods commonly utilized for miRNA expression studies yield highly unstable miRNAs. We studied the stability of four miRNAs belonging to different miRNAs families. A significant degradation of these molecules may be observed already three days after RNA isolation. Moreover, the respective cDNAs are highly unstable as well. Our findings indicate that instability of miRNAs and their cDNAs should be considered when designing miRNA expression studies.