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RNA Spike-In Kit, For RT

For quality control of the RNA isolation and cDNA synthesis steps of miRCURY LNA miRNA PCR experiments
  • An easy and convenient control of your RNA samples and RT reactions
  • Save time and reagents by identifying poor-quality samples early
  • Available in an easy-to-use, pre-mixed format
  • Highly flexible – use only the spike-ins you need for your study

The RNA Spike-In Kit enables quality control of the RNA isolation, cDNA synthesis and PCR amplification steps of miRCURY LNA miRNA qPCR experiments. Identifying any poor-quality samples early in the process saves you time and reagents. Plus, the kit is easy to use and comes in a premixed format that allows you to use only the spike-ins you need for your analyses.

Cat No./ID: 339390
RNA Spike-In Kit, For RT
$246.00
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Contains the UniSp2, UniSp4, and UniSp5 RNA Spike-in Template Mix and the cel-miR-39-3p RNA Spike-in Template
The RNA Spike-In Kit, For RT is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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miRCURY LNA miRNA QC PCR Panels can be used with a wide range of sample types.
The miRNAs on the panel are expressed in many sample types. In this example, human serum, plasma, urine, colorectal cancer (FFPE) and mouse brain (fresh frozen) samples were analyzed.
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Determine RNA yields using the RNA Spike-in Kit.
miRCURY LNA miRNA QC PCR Panels offer a convenient way of determining RNA yields. In this example, a plasma sample was purified 5 times using three different RNA isolation methods. RNA spike-ins from the RNA Spike-in Kit were added to the lysis buffers of the isolation kits according to the kit handbook. RNA isolation B gives the highest yield, as indicated by the lower Cq values. Overall PCR efficiency is similar between the methods, as determined by the similar UniSp3 results. The high Cq value of UniSp6 from isolation A could indicate the presence of a PCR inhibitor in this sample.
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Assessing if samples are affected by hemolysis.
The data from the red blood cell-specific miR-451 and the stable miR-23a can be used to monitor hemolysis. Shown is the difference in the level (ΔCq) of miR-23a and miR-451 for 34 plasma samples. A ΔCq(miR-23a – miR-451) lower than 5 in human serum or plasma represents non-hemolyzed samples. If the ΔCq is close to or higher than 7, there is an increased risk that the samples are affected by hemolysis. In case of high levels of hemolysis, miRNAs from red blood cells will make a significant contribution to the overall miRNA profile identified. Special care must be taken in the analysis of such samples
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The RNA Spike-in Kit, for RT covers the full range of miRNA expression levels.
The RNA Spike-In Kit, for RT was used for qPCR quality control of commercially available plasma from two different sources (n=2 for each). The UniSp2 / UniSp4 / UniSP5 RNA Spike-in mix was introduced in the RNA isolation step (blue), and the UniSp6 / cel-miR-39-3p RNA Spike-in mix was added to the RT reaction (red), as described in the kit handbook. As seen from the Cq values, the kit offers a wide dynamic range, which is ideal for control of both the RNA isolation and RT reaction steps Moreover, the results show that the RNA isolation and the RT reaction were highly reproducible.
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Overview of issues and conclusions.
Performance
Checking sample quality and PCR efficiency
Together with the miRCURY LNA miRNA QC PCR Panel, the RNA Spike-in Kit for RT offers a unique and comprehensive way of checking sample quality and PCR efficiency. The panel is compatible with many sample types, including cell cultures, tissue samples and biofluids from human, mouse, rat, monkey and dog (see figure miRCURY LNA miRNA QC PCR Panels can be used with a wide range of sample types).

Test your isolated RNA to find outliers and suboptimal samples (see figure Determine RNA yields using the RNA Spike-in Kit), including serum/plasma samples affected by hemolysis (see figure Assessing if samples are affected by hemolysis). This saves time and resources in the laboratory.
Principle
Features
Use the RNA Spike-In Kit for quality control of the RNA isolation and cDNA synthesis steps of your miRCURY LNA miRNA PCR experiments. It is important for any qPCR experiment to ensure that the quality of the input RNA is sufficiently high for effective amplification. By introducing spike-in RNAs during RNA isolation and cDNA synthesis and comparing the qPCR results to those from endogenous reference genes, you can gain important information about the quality of your samples (see figure Overview of issues and conclusions). The kit offers a wide dynamic range, which is ideal for controlling these processes (see figure The RNA Spike-in Kit covers the full range of miRNA expression levels).

Product coverage
Our RNA spike-ins (UniSp2, UniSp4, UniSp5, UniSp6) resemble miRNAs in structure but lack close sequence similarities to known miRNAs. The kit also includes a synthetic version of the C. elegans miRNA, cel-miR-39-3p.

The RNA Spike-In Kit is distributed in two vials:
  • Vial 1 contains 3 RNA spike-ins (UniSp2, UniSp4 and UniSp5) pre-mixed, each at different concentration in 10-fold increments (see table below). This set of spike-ins is intended as an RNA isolation control.
  • Vial 2 contains a synthetic version of cel-miR-39-3p. This miRNA is meant to be used in conjunction with the UniSp6 spike-in provided with the miRCURY LNA RT Kit (denoted "RNA spike-in"). Mixing these RNA templates as described in the kit handbook will result in a 100-fold concentration difference between the RNAs (see table below). This set of spike-ins is intended as a cDNA synthesis control.

Each vial contains RNA sufficient for 50 reactions. Also included is MS2 carrier RNA, which improves the stability of the RNA spike-ins without interfering with downstream reactions.

Contents of the RNA Spike-In Kit
Reagent Amount Conc. (resuspended)
UniSp2, UniSp4, UniSp5 RNA Spike-in template mix:
Synthetic UniSp2 RNA (22 nt) 160 fmol 2 fmole/µl
  Synthetic UniSp4 RNA (22 nt) 1.6 fmol 0.02 fmole/µl
  Synthetic UniSp5 RNA (22 nt) 0.016 fmol 0.00002 fmole/µl
  MS2 total RNA 50 ng 0.625 ng/µl
cel-miR-39-3p RNA Spike-in template:
Synthetic cel-miR-39-3p RNA 0.16 fmol 0.002 fmole/µl
  MS2 total RNA 50 ng 0.625 ng/µl


Choose the spike-in RNAs you prefer
To take full advantage of the RNA Spike-In Kit, you will need to purchase the corresponding LNA primer sets separately, with the exception of UniSp6, which is included in the miRCURY SYBR Green PCR Kit. This gives you the flexibility to use only the spike-ins you need without having to pay extra for primer sets you are not going to use.

Compatibility
The RNA Spike-In Kit is compatible with the miRCURY LNA miRNA PCR Assays and Panels.
Procedure
Analysis and interpretation of data
An important factor when using the RNA Spike-In Kit is knowing how to interpret the experimental data. We have made this easy by devoting an entire section in the kit handbook to explaining how to analyze and interpret the data in an easy-to-understand and clear-cut way. Refer to the RNA Spike-In Kit Handbook for details.
Applications
Quality control of the RNA isolation and cDNA synthesis steps of miRCURY LNA miRNA PCR experiments
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