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QIAseq Targeted RNA Indexes

用于对目标RNA测序所需样本和对QIAseq Targeted RNA Panels生成的RNA文库测序进行索引
  • 为Illumina和Ion Torrent测序仪提供最多96个样本的索引
  • 每个索引可足够用于4份样本
  • 管式包装具有灵活性,板式包装符合便利性和高通量测定的要求

 

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Cat No./ID: 333114
QIAseq Targeted RNA 12-Index I (48)
Kit containing sample indexes, enough for a total of 48 samples, for indexing up to 12 samples for targeted RNA sequencing on Illumina platforms (tube format), and primers necessary for sequencing RNA libraries generated by the QIAseq Targeted RNA Panels on Illumina platforms
Cat No./ID: 333117
QIAseq Targeted RNA 96-Index I (384)

Kit containing sample indexes, enough for a total of 384 samples, for indexing up to 96 samples for targeted RNA sequencing on Illumina platforms (tube format), and primers necessary for sequencing RNA libraries generated by the QIAseq Targeted RNA Panels on Illumina platforms

Cat No./ID: 333127
QIAseq Targeted RNA 96-Index HT l (384)

Kit containing sample indexes, enough for a total of 384 samples, for indexing up to 96 samples for targeted RNA sequencing on Illumina platforms (array format), and primers necessary for sequencing RNA libraries generated by the QIAseq targeted RNA panels on Illumina platforms

Cat No./ID: 333214
QIAseq Targeted RNA 12-Index L (48)
Kit containing sample indexes, enough for a total of 48 samples, for indexing up to 12 samples for targeted RNA sequencing on Ion Torrent platforms (tube format)
Cat No./ID: 333217
QIAseq Targeted RNA 96-Index HT L (384)

Kit containing sample indexes, enough for a total of 384 samples, for indexing up to 96 samples for targeted RNA sequencing on Ion Torrent platforms (array format)

QIAseq Targeted RNA Indexes适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


Product Details

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One solution to overcome the challenges of gene expression profiling

The QIAseq Targeted RNA Panels have been developed as a Sample to Insight solution for quantitative gene expression profiling using RNAseq. The panels use molecular barcodes and a two-stage PCR-based integrated library preparation to overcome the challenge of PCR duplicates and amplification bias to deliver unbiased, accurate and reproducible gene expression results.

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High concordance with qPCR
Expression levels for 384 genes were determined by both QIAseq Targeted RNA Panel and qPCR for Human Brain Reference RNA (HBRR) and Universal Human Reference RNA (UHRR) samples. The expression levels determined by qPCR were normalized to the average of four housekeeping genes (ACTB, B2M, GAPDH and RPLP0) and fold change between the samples was calculated (HBRR/UHRR) for each gene. For QIAseq, the number of unique molecular barcodes per gene were counted and normalized to average number of molecular barcodes for the four housekeeping genes for each sample. The fold change for each gene was then calculated. The QIAseq Targeted RNA Panel and qPCR assays exhibit similar fold-changes in gene expression, highlighting the accuracy of QIAseq-results.
9
Proprietary primer design delivers gene-specific amplicons (>97% specificity)

Sequencing libraries were prepared using 1.25, 5 or 20 ng universal reference RNA and QIAseq Targeted RNA Panels, ranging from 12-plex to 1000-plex. Sequencing was performed on the Illumina MiSeq, dedicating 1 million reads per sample. Specificity is calculated as percent of trimmed and mapped reads that map to intended targets.

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Simple procedure

Starting with 25 ng of total unfragmented RNA, cDNA is synthesized, and each cDNA molecule is tagged with a unique molecular barcode before any amplification. The uniquely tagged cDNA molecules then undergo a two-stage PCR step for enrichment and library construction. It takes only 6 hours from RNA sample to targeted library ready for sequencing.

9
Unmatched uniformity (>97% of assays are within 20% of median molecular tag counts)
A 917-plex gene panel was used to prepare a library from 10 ng of NA12878 reference DNA. All assays were designed to be intra-exon, and are thus single copy on genomic DNA. This allows an estimation of the uniformity of amplicon performance in the library preparation step (e.g., every unique tag equals one captured copy). In terms of raw assay performance, 97.5% of assays are within 20% of mean/median molecular tag counts. For cataloged panels, any assay below 20% is redesigned and replaced. Molecular barcodes entirely remove this variation in RNAseq counting.
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Unbiased and accurate gene quantification (B)
Different amounts (20 ng, 5 ng or 1.25 ng) of universal reference RNA were used to determine expression levels of three genes (BNIP1, CTNND2 and DAPK1) using targeted RNAseq. QIAseq digital RNA sequencing method (A; molecular barcode counts) showed accurate quantification of all three genes corresponding to different RNA input, whereas traditional targeted RNAseq (B; read counts) revealed PCR duplication limitation and yielded inaccurate quantification.
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Unbiased and accurate gene quantification (A)

Different amounts (20 ng, 5 ng or 1.25 ng) of universal reference RNA were used to determine expression levels of three genes (BNIP1, CTNND2 and DAPK1) using targeted RNAseq. QIAseq digital RNA sequencing method (A; molecular barcode counts) showed accurate quantification of all three genes corresponding to different RNA input, whereas traditional targeted RNAseq (B; read counts) revealed PCR duplication limitation and yielded inaccurate quantification.

9
Digital sequencing (molecular barcodes) principle

QIAseq Targeted RNA Panels use a digital sequencing method, whereby a unique 12-base random molecular barcode incorporated into the gene-specific primers (GSP1) is used in the first extension step (after mRNA is converted to cDNA). Thus, every extension event yields a unique combination of molecular barcode and target sequence. At the end of sequencing, the relative amount of each mRNA target is determined by the number of unique molecular barcode-target combinations that were sequenced, thereby eliminating PCR duplicates and amplification bias, resulting in more accurate, unbiased gene expression analysis.

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Positive results with as little as 0.2 copies of RNA per cell
ERCC standards, at 86 to 705,500 copies, spiked into universal reference RNA sample and enriched using 384-plex QIASeq Targeted RNA Panel in three technical replicates. (A) Sensitivity measurement. Under standard conditions (20 ng RNA input, 0.5 million MiSeq reads), ≥~100 copies of ERCC transcripts were reliability detected, which is the equivalent of ~0.2 copies per cell. (B) Precision measurement. At >10 barcodes/gene, CV was less than 5% for all targets, indicating high technical reproducibility. This corresponds to ~100 copies target RNA in the sample.
Performance
  • 准确性:创新的数字测序技术(分子条形码计数法)消除了PCR复制和扩增引入的偏差,可获得准确的结果(参见图无偏差、准确的基因定量检测结果)。
  • 特异性:独创性地将专有的引物设计算法和每个引物延伸实验的严格检测相结合,保证了结果的高度特异性和准确性(参见图专有的引物设计– 97%的特异性)。
  • 一致性:QIAseq Targeted RNA Panel工作流程已经过优化,可获得高度一致的测序结果,保证仪器的测序能力得到有效利用。实际上,数字测序(分子条形码)已完全消除了RNAseq计数中产生的数据波动(请参见图卓越的一致性 – 97%的实验结果均处于分子标记数中位数的20%内)。
  • 可重复性:QIASeq Targeted RNA Panel系统重复性强,在重复样本、不同批次产品和不同仪器的检测结果间存在很强的相关性,平均相关系数高于0.99,保证了检测的可靠性。
  • 灵敏性:数字RNA测序系统已经过优化,可获得极为可靠的定量检测结果,可定量25 ng总RNA之中低至100个拷贝的RNA靶标(参见图每个细胞低至0.2个拷贝数RNA的阳性测定结果)。
  • 灵活性:QIAseq panel将NGS的强大功能和qPCR的准确性合为一体。在获得高性价比数据结果的同时,可通过单次NGS多重检测多个样本。
Principle
  • 传统的RNA测序方法会受到PCR反应中模板复制和扩增的影响而引入误差,导致基因表达分析不够准确。QIAseq Targeted RNA Panel在进行PCR扩增反应之前引入分子条形码,消除了上述问题从而能够对基因进行准确的数字化定量(参见图客观、准确的基因定量)。
  • QIAseq Targeted RNA Panel的优势在于包含内参反应体系,能够对RNA样本中任何由gDNA造成的污染进行控制,从而获得可重复性高的实验结果。管家基因(HKG)实验用于归一化数据,从而可对比各样本及运行批次间的结果。
Procedure
  • QIAseq Targeted RNA Panel在使用时,首先将所有的RNA全部逆转录成cDNA(参见图简单的操作流程)。实验所需的起始总RNA量可低至25 ng,而无需进行任何富集步骤。在分子条形码标记步骤,需要使用多重检测引物panel(靶向12-1000个基因)中带有分子标签且具有基因特异性的引物(GSP1),和20 ng的cDNA等效物(由20ng RNA逆转录得到的cDNA)。在条形码标记步骤之后,将带有特异标记的cDNA用磁珠纯化以去除残留的引物。随后,采用第二个引物池构建PCR。该引物池中含有带基因特异性接头的引物(GSP2)和可去除GSP1引物通用标签的RS2引物。该反应可确保预定目标基因得到充分富集从而出现在最终的文库中。此设计可以最大限度缩短反应循环次数,最大限度减少扩增过程中PCR引入的误差(所有误差均可用分子标签作简单修正)。第二次反应之后同样用磁珠对cDNA进行纯化并采用RS2和FS2引物进行universal PCR,反应中对每个样本同样加入样本索引条形码,以用于测序过程中的样本区分。纯化最终产物后,即构建出可直接用于定量和测序的文库。
  • QIAseq Targeted RNA Panel的功能包括数据分析和解读。目前我们已为其开发出全面、易用的数据分析模块。即使您并非生物信息学专家,也可自如地使用这些模块。首先从测序仪上获得原始序列,再使用QIAGEN的GeneGlobe网站首页上的QIAseq靶向RNA数据分析工具,即可获得基因数量、倍数变化的信息以及通路分析结果。
Applications
  • 生物标志物研究
  • 全转录组测序数据验证
  • 微阵列数据验证

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