Quant-iT™ RiboGreen™ RNA Assay Kit, 1 kit - FAQs

为什么我用Qubit Assay检测时会得到负的荧光值？

负的荧光值物理上是不可能的。它是由于软件自动扣除背景信号而造成的假象。这意味着你的荧光计检测到背景信号并将其扣除从而牺牲了真实数据。务必做一个仅有缓冲液的对照并评估信号的类型。你可能需要换用另外一块板。

Quant-iT RNA试剂盒的线性范围是多少？

线性范围如下：

•Quant-iT RiboGreen RNA检测试剂：1 ng/mL至1 µg/mL
•Quant-iT RNA BR检测试剂：20–1,000 ng
•Quant-iT RNA HS检测试剂：5–100 ng

Quant-iT RNA检测试剂和Quant-iT RiboGreen RNA检测试剂之间有什么区别？

有以下一些区别：

•Quant-iT RNA检测试剂使用一个远红外染料（激发光644nm/发射光673nm）。
Quant-iT RiboGreen RNA检测法使用一个绿色荧光染料（激发光500nm/发射光525nm）。
•Quant-iT RNA检测试剂对RNA更具特异性。
•Quant-iT RNA检测试剂比Quant-iT RiboGreen RNA检测试剂灵敏度低

我们可以为Qubit荧光计制定我自己的检测方案吗？

是的，对于Qubit (1.0)荧光计之后的Qubit设备是可以的。点击此处（https://www.thermofisher.com/cn/zh/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-assays/myqubit.html）查看MyQubit检测说明。

我有一个粗裂解物。使用Quant-iT和Qubit检测试剂能行吗？

通常来说，样本越干净越好。一些盐、蛋白、以及去垢剂不会影响检测，您可以查看特定的检测方案以了解哪些物质以及它们在哪些浓度下不会影响检测。

Why am I getting negative fluorescence values with my Qubit Assays?

Negative fluorescence is a physical impossibility. It is an artifact from software autocorrecting for background signal. This means your reader is picking up and subtracting out background light at the cost of your data. Make sure to do a buffer-only control and assess the type of signal. You may need to switch to a different plate.

What are the linear ranges of the Quant-iT RNA kits?

The linear ranges are:

- Quant-iT RiboGreen RNA Assay: 1 ng/mL to 1 µg/mL
- Quant-iT RNA BR Assay: 20-1,000 ng
- Quant-iT RNA HS Assay: 5-100 ng

What is the difference between the Quant-iT RNA assay and the Quant-iT RiboGreen RNA Assay?

There are several differences:

- The Quant-iT RNA Assay uses a far-red dye (excitation 644 nm/emission 673 nm); the Quant-iT RiboGreen RNA assay uses a green fluorescent dye (excitation 500 nm/emission 525 nm).
- The Quant-iT RNA assay is more specific for RNA.
- Quant-iT RNA Assays are less sensitive than the Quant-iT RiboGreen RNA Assay.

Can I make my own assay for the Qubit Fluorometer?

Yes, you can, for Qubit instruments developed after the original Qubit (1.0) Fluorometer. See MyQubit assay instructions here (http://www.thermofisher.com/us/en/home/life-science/laboratory-instruments/fluorometers/qubit/qubit-assays/myqubit.html.html).

I have a crude lysate. Will the Quant-iT and Qubit assays work?

Generally, the cleaner the sample the better. Some salts, proteins, and detergents are tolerated in the assays; see the specific assay protocol for which ones and at what concentrations.

How does the accuracy and sensitivity of the Qubit quantitation assays using the Qubit fluorometer compare to a microplate reader?

The accuracy and sensitivity of the Qubit quantitation assays are the same as that of a microplate reader. This was a requirement during product development. The detection limits for each Qubit kit can be found on the corresponding product manual, which can be found by searching our website by keyword or catalog number.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.

Can the Qubit kits give an indication of sample quality in nucleic acid samples?

No. The Qubit DNA and RNA kits only quantify the amount of either DNA or RNA in the sample. The Qubit fluorometer cannot take absorbance readings to provide a A260/A280 ratio or detect protein in nucleic acid samples. This can be done with the NanoDrop instrument. If your sample contains protein or other contaminants that can affect the assay, it should be further purified.

If your sample may contain both DNA and RNA, one may use either (or both) the DNA and RNA Qubit kits and compare with samples treated with either RNase or DNase to get an accurate determination of DNA or RNA, respectively.

Can I use the Quant-iT and Qubit Kits with other fluorometers?

All Quant-iT and Qubit kits are compatible with all fluorometers and microplate readers that have the appropriate light sources and filters. You won't have access to the algorithm in the Qubit fluorometer for generating the standard curve provided by the instrument, instead, you must make a few dilutions of the highest standard DNA or RNA (Standard #2) in the Qubit kits to generate a standard curve with multiple data points.

Can I use the original Quant-iT Kits with the Qubit Fluorometer?

No, we do not recommend this. Some of the dyes in the original Quant-iT kits (those NOT listed as “for use with the Qubit fluorometer”) are not compatible with the Qubit Fluorometer. In addition, the new Quant-iT kits (labeled as “for use with the Qubit Fluorometer”) have standards formulated to be compatible with the Qubit fluorometer internal algorithms for the respective assays. The Qubit Fluorometer-compatible kits are also less expensive per assay if you are processing fewer than 20 samples at a time.

My Quant-iT RiboGreen RNA Kit (Cat. No. R11490) was shipped at ambient temperature but the recommended storage is 2-8 degrees C. Will this affect the stability or performance?

The Quant-iT Ribogreen RNA Kit (Cat. No. R11490) is routinely shipped at ambient temperature. Functional and analytic testing has shown that shipping this product at ambient temperature provides the same product quality as shipping the product on gel ice without impacting long-term stability or performance. The Quant-iT RiboGreen RNA Reagent (Component A) and Ribosomal RNA standard 16S and 23S rRNA (Component C) should be stored at 2-8 degrees C as directed in the User Guide upon receipt.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.