Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.
Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.
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DOI：
10.1186/s12867-015-0039-3
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发表时间：
2015-05-06
影响因子：
--
通讯作者：
Williams Z
中科院分区：
文献类型：
--
作者：
Li X;Ben-Dov IZ;Mauro M;Williams Z
RNA定量通常是大多数RNA分析（如RNA测序）的先决条件。然而，传统的RNA定量方法，如紫外分光光度法，灵敏度相对较低，样品消耗大，甚至更敏感的基于荧光的RNA定量测定，如Qubit™RNA HS测定，往往不足以测量从有限的细胞和组织样品和生物液体中分离的微量RNA。因此，迫切需要一种更灵敏的方法来可靠和健壮地检测痕量RNA，而不受DNA的干扰。为了提高Qubit™RNA HS测定的定量极限，我们加入了已知数量的RNA以达到测定所需的最小读数。然后将含有痕量RNA的样品添加到峰值中，并测量读数在RNA峰值基线上的增加。我们测定了读数在1 ~ 20 pg/μL之间的准确度和精密度以及在此范围内的RNA特异性，并与另一种敏感的基于荧光的RNA定量测定方法RiboGreen®进行了比较。然后，我们应用带有RNA峰入的Qubit™Assay来定量血浆RNA样品。RNA峰入将Qubit™RNA HS Assay的定量限提高了5倍，从25 pg/μL降至5 pg/μL，同时保持对RNA的高特异性。这使得原始浓度低至55.6 pg/μL的RNA定量，而标准测定为250 pg/μL，样品消耗从5减少到1 ng。不能用Qubit™RNA HS Assay测量的血浆RNA样品可以用我们改进的方法测量。与标准Qubit™检测相比，具有RNA峰值的Qubit™RNA HS检测能够以低5倍的浓度以高特异性定量RNA，并且使用的样品量少5倍。本文的在线版本（doi:10.1186/s12867-015-0039-3）包含补充材料，授权用户可以使用。
RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen®, another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay. The online version of this article (doi:10.1186/s12867-015-0039-3) contains supplementary material, which is available to authorized users.
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影响因子：
2.9
作者：
RYE, HS;DABORA, JM;GLAZER, AN
通讯作者：
GLAZER, AN
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
2.9
作者：
LABARCA, C;PAIGEN, K
通讯作者：
PAIGEN, K
DOI：
10.1186/1758-907x-1-7
发表时间：
2010-03-01
期刊：
Silence
影响因子：
--
作者：
Kosaka N;Izumi H;Sekine K;Ochiya T
通讯作者：
Ochiya T
影响因子：
4.5
作者：
Burgos, Kasandra Lovette;Javaherian, Ashkan;Van Keuren-Jensen, Kendall
通讯作者：
Van Keuren-Jensen, Kendall