Zhiyao Chen

Dye‐Free MicroRNA Quantification by Using Pyrosequencing with a Sequence‐Tagged Stem–loop RT Primer

MicroRNAs (miRNAs) are a class of endogenous, ~22-nucleotide (nt) noncoding RNAs that play an important role in the control of the developmental processes of cells by negative regulation of protein-coding gene expression. To date, there are 17 341 mature miRNAs, including 1048 human miRNAs, in the University of Manchester miRNA database (http://www. mirbase.org/). Although miRNAs represent a relatively abundant class of transcripts, their expression levels vary greatly in different tissue types and species. Analyzing miRNA expression levels in tissues or cells can supply valuable information for investigating the biological functions of miRNAs; however conventional techniques to amplify miRNAs for detection and quantification present a significant challenge because of the short length of these molecules; thus, a number of straightforward methods without the use of amplification have been developed for miRNA detection. Northern blotting 5] is the widely used standard method for analyzing miRNAs; however, relatively large amounts of starting material (RNA) are required for an assay. To improve the sensitivity of miRNA quantification, a method based on splinted ligation was developed. This exhibits approximately 50 times greater sensitivity than Northern blotting, but radioactive P labels are needed. A single-molecule method, based on the hybridization of two spectrally distinguishable LNA–DNA oligonucleotide probes (for the miRNA of interest), offers a direct miRNA assay as sensitive as 500 fm, but an expensive single-molecule detection instrument is required. For sensitive miRNA detection, amplification techniques are thus necessary. By skillfully designing detection probes, a modified “Invader” assay was developed for the quantification of miRNAs. Although 20 000 miRNAs were detected, accurate quantification of miRNAs among samples is difficult because the initial target concentration is proportional to the steady-state reaction rate of “invasive” amplification. In contrast, an miRNA assay based on real-time quantitative PCR with a stem–loop reverse transcription (RT) primer was much more quantitative, as the Ct (cycle threshold) value is inversely proportional to the amount of initial target. However, PCRs of the sample and reference targets are performed separately, and a small difference in amplification efficiency between the sample and the reference yields a large difference in the amount of final product ; this results in large inter-PCR variations. Recently, a simple and sensitive miRNA quantification method that used branched rolling-circle amplification (BRCA) was reported, but quantification based on endpoint readout seems challengeable because of the time-dependent amplification efficiency of BRCA. To achieve accurate quantification of a target miRNA in a sample, real-time monitoring of signal intensities from both a sample and a reference (quantification standard) is necessary, because the reaction rate slows down as the reaction proceeds. As the real-time detection requires a sophisticated instrument, quantification using endpoint data is preferable. In the present study, we have developed a pyrosequencing-based method for absolute quantification, and for comparing the relative miRNA expression levels in biological samples. Pyrosequencing is a well-developed technology for DNA sequencing. It uses cascade enzymatic reactions to monitor the release of inorganic pyrophosphate that results from dNTP incorporation. Because of its highly quantitative performance, pyrosequencing has been widely used for genotyping, and the analysis of DNA methylation and gene expression. Here we employed pyrosequencing technology to quantify microRNAs by quantitatively detecting sequence labels that were artificially tagged into the RT products of miRNA. Unlike mRNA, miRNA is very short and can be easily synthesized; synthesized molecules with known concentration could thus be used as a reference for quantifying miRNA in a sample. As shown in Figure 1, sequence labels for discriminating the sources of miRNA (sample or reference) are designed into the loop near to the 3’-end of the miRNA-specific RT primer, so that the 5’ end of the primer can offer a universal priming site for the following PCR. The structure of the miRNA-specific stem–loop RT primer is the same as that used by Chen’s group. After reverse transcription with the sequence-tagged RT primers, cDNA from the different sources (sample and reference) were similarly labeled with different sequences (thus, different colors in a fluorescence-based assay). To avoid PCR-bias resulting from Tm differences, the labels were designed from the same base species but with different base order. We labeled the sample-miRNA and the reference-miRNA with the sequences “catg” and “gatc” respectively ; hence, in a pyrogram (Figure 1), [a] H. Jing, Prof. Q. Song, Z. Chen, B. Zou, Prof. G. Zhou Huadong Research Institute for Medicine and Biotechnics Nanjing 210002 (China) Fax: (+ 86) 25-8451-4223 E-mail : ghzhou@nju.edu.cn [b] Prof. Q. Song, B. Zou School of Life Science and Technology, China Pharmaceutical University Nanjing 210009 (China9 [c] C. Chen, Prof. M. Zhu Model Animal Research Centre, Nanjing University Nanjing 210093 (China) [d] Z. Chen, Prof. G. Zhou Medical School, Nanjing University Nanjing 210093 (China) [e] T. Kajiyama, Prof. H. Kambara Central Research Laboratory, Hitachi, Ltd. Tokyo 185-8601 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100023.

Pyrosequencing-based barcodes for a dye-free multiplex bioassay

A novel dye-free labeling method for a multiplex bioassay was proposed by using short sequence-based barcodes consisting of a reporter base and repeats of two stuffer bases; then, the barcodes were quantitatively decoded by a single pyrosequencing assay without any pre-separation.

Preparation of Single-Stranded DNA for Pyrosequencing by Linear-after-the-Exponential-Polymerase Chain Reaction

Abstract To establish a simple method for preparing single-stranded DNA templates for pyrosequencing, the Linear-after-the-exponential (LATE)-PCR technology on the basis of Taq DNA polymerase without hot-start capacity was applied to amplify a 78-bp sequence (containing the SNP6 site), and the PCR-enhancing reagents (glycerol and BSA) were used to increase the efficiency and specialization, much more before the reagents A and B were designed to eliminate the impurity (limited primers, PPi, dNTPs and so on), and 1–2 μl LATE-PCR products with simply treatment can be used in pyrosequencing directly. Then, five SNPs related with human breast-cancers in the BRCA1 gene were investigated, and the programs had no nonspecific signals that were made of theoretic sequences. Moreover, the genotyping of the SNPs could also be distinguished easily. The results indicated that this method can be used to prepare high quality single-stranded DNA templates for pyrosequencing and allows pyrosequencing be lower in cost, simpler in operation, and easier in automation, and the cross-contamination from sample preparation was also reduced.

Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing.

Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.

A simplified pyrosequencing protocol based on linear-after-the-exponential (LATE)-PCR using whole blood as the starting material directly

Pyrosequencing has been one of the most commonly used methods for genotyping; however, generally it needs single-stranded DNA (ssDNA) preparation from PCR amplicons as well as purified genomic DNA extraction from whole blood. To simplify the process of a pyrosequencing protocol, we proposed an improved linear-after-the-exponential (LATE)-PCR by employing whole blood as the starting material. A successful LATE-PCR was achieved by using a common Taq DNA polymerase in high pH buffer (HpH-buffer). As amplicons from LATE-PCR contain a large amount of ssDNA, pyrosequencing can be performed on the amplicons directly. Since DNA extraction and ssDNA preparation are omitted, the labor, cost and cross-contamination risk is decreased compared to conventional pyrosequencing-based genotyping protocols. The results for typing three polymorphisms related to personalized medicine of fluorouracil indicate that the proposed whole-blood LATE-PCR can be well coupled with pyrosequencing, thus becoming a potential tool in personalized medicine.

A Simplified Protocol for Preparing Pyrosequencing Templates Based on LATE-PCR Using Whole Blood as Starting Material Directly

Pyrosequencing has been one of the most commonly used methods for genotyping; however, generally it needs single-stranded DNA (ssDNA) preparation from PCR amplicons as well as purifi ed genomic DNA extraction from whole blood. To simplify the process of a pyrosequencing protocol, we proposed an improved linear-after-the-exponential (LATE)-PCR by employing whole blood as starting material. A successful LATE-PCR was achieved by using a common Taq DNA polymerase in high pH buffer (HpHbuffer). As amplicons from LATE-PCR contain a large amount of ssDNA, pyrosequencing can be performed on the amplicons directly. Since DNA extraction and ssDNA preparation are omitted, the labor, cost, and cross-contamination risk is decreased comparing to conventional pyrosequencing-based genotyping protocols. The results for typing three polymorphisms related to personalized medicine of fl uorouracil indicate that the proposed whole-blood LATE-PCR can be well coupled with pyrosequencing, thus becoming a potential tool in personalized medicine.

Differential Gene Expression Analysis by Combining Sequence-Tagged Reverse-Transcription Polymerase Chain Reaction with Pyrosequencing

Abstract It is an important way to understand the gene function by relatively comparing gene expression levels among different tissues or cells. For the moment, most of the methods for gene expression detection are based on dye labels. To establish a novel approach without using a dye label, a sequence-tagged reverse-transcription PCR coupled with pyrosequencing (SRPP) was proposed. In this technique, the gene from a source is labeled with a source-specific sequence by sequence-tagged reverse transcription (RT). Then PCR on the pools of each source-specific RT product was performed, and the source-specific amplicons were decoded by pyrosequencing. In the pyrogram, the sequence represents the gene source, and the peak intensity represents the relative expression level of the gene in the corresponding source. The accuracy of SRPP was confirmed by real-time quantitative PCR. Finally, the relative expression levels of the Egr1 gene among the diabetes model mice, obesity model mice, and normal mice were successfully detected. In comparison with real-time quantitative PCR, the advantages of SRPP include dye-free detection, inexpensive instruments, and simultaneous comparison of a given gene expressed in multiple sources.