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首页> 外文期刊>BMC Molecular Biology >Lowering the quantification limit of the Qubit TM RNA HS Assay using RNA spike-in
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Lowering the quantification limit of the Qubit TM RNA HS Assay using RNA spike-in

机译：使用RNA掺入降低Qubit TM RNA HS分析的定量限

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摘要

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.
机译：RNA定量通常是大多数RNA分析（例如RNA测序）的先决条件。但是，传统的RNA定量方法（例如紫外分光光度法）以及相对敏感得多的基于荧光的RNA定量分析（例如Qubit™RNA HS分析）相对较低的灵敏度和较大的样品消耗量通常不足以测量微小的RNA水平分离自有限的细胞和组织样品以及生物流体。因此，迫切需要一种更灵敏的方法来可靠而可靠地检测痕量的RNA，而不会受到DNA的干扰。为了提高Qubit™RNA HS分析的定量极限，我们掺入了已知数量的RNA，以达到分析所需的最小读数。然后将包含痕量RNA的样品添加到尖峰中，并以相对于RNA尖峰基线的读数增加进行测量。我们确定了在1至20 pg /μL之间增加读数的准确性和精确度以及该范围内的RNA特异性，并将其与另一种基于荧光的敏感RNA定量测定RiboGreen®进行了比较。然后，我们将Qubit™分析与RNA掺入法一起用于定量血浆RNA样品。 RNA刺入将Qubit™RNA HS分析的定量限提高了5倍，从25pg /μL降至5pg /μL，同时保持了对RNA的高特异性。与标准检测方法中250 pg /μL的浓度相比，原始浓度低至55.6 pg /μL的RNA定量功能使样品消耗量从5 ng减少至1 ng。 Qubit™RNA HS分析无法测量的血浆RNA样品可通过我们的改良方法进行测量。带有RNA插入功能的Qubit™RNA HS分析能够以低5倍的浓度高特异性定量RNA，所用样品量比标准Qubit™分析低5倍。

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