EVery miRNA Spike-in Kit

Product Description

Get a better control over your miRNAs research with EVery miRNA Spike-in Kit which can help you assess the efficiency of your RNA isolation and cDNA synthesis.

• Separate Spike-in mixes for RNA isolation and cDNA synthesis steps
• Suitable for miRNA from any source or method of isolation
• Cover high, medium and low abundance miRNAs
• Forward primer supplied for qPCR

Quality and integrity of RNA as well as efficiency of isolation methods is critical to maintain the representation of any transcriptome. Because miRNAs are very short RNA species, they may behave very differently during the isolation and reverse transcription process than the longer RNA transcripts. Therefore, it is very important to have controls that resemble endogenous miRNAs closely. Synthetic RNAs that resemble the size of endogenous miRNAs but do not share any homology with any identified miRNAs are ideal spike-ins. Optimized mix of such spike-ins offers a control for endogenous miRNAs of low, medium and high abundance levels reflecting closely the fidelity or impact of any RNA isolation methods. The RNA Spike-ins Spkn1, Spkn2 and Spkn3 are at a 100-fold concentration difference, therefore, there should be ~6.6 Ct difference between the serial dilutions of spike-ins. The concentration of Spkn1 corresponds to very abundant miRNAs, and the concentration of Spkn3 corresponds to weakly expressed miRNAs and might not always be detectable. Undetectable Spkn3 could be an indication that miRNAs expressed at low levels were lost during isolation. A separate Spike-in mix (Spkn4 and cel-miR-39-3p) added later during cDNA synthesis reports on the efficiency of reverse transcription and PCR amplification that may be affected by inhibitors.

How it Works

Using EVery miRNA Spike-in Kit

1. Use them as directed to spike-in your sample during RNA isolation (Spkn1, Spkn2 and Spkn3 mix) and cDNA synthesis (Spkn4 and cel-miR-39-3p mix).
2. Then conduct qPCR assays using the forward primers supplied for the Spike-ins
3. Interpret the data based on the table supplied in the user manual.
   
   

Supporting Data

Expression of spike-in miRNAs in serum and plasma EV samples

To demonstrate the excellent EV RNA yields and robust cDNA synthesis, we isolated EVs from 250 µL of serum or plasma using SmartSEC Single, spiked in with our synthetic EVery miRNA Spike-in mix, and used the EVery EV RNA Isolation Kit and the EVery cDNA Synthesis Kit. Examples of RNA spike-ins isolated with serum and plasma samples are shown in Figure 1.

Figure 1. EVery miRNA Spike-in Kit reflects successful miRNA isolation. EV RNA from human serum and plasma samples were isolated with EVery family of products. The synthetic RNA spike-in mixture was added during sample preparation stepwise according to EVery miRNA Spike-in Kit user manual. The RNA spike-in yields can be monitored with Spkn1, 2, and 3; the potential presence of inhibitors can be monitored with cel-miR-39 and Spkn4. The stepwise difference in Ct values between the RNA isolation spike-ins (Spkn1, 2, 3) was in the expected range of 5-7. Error bars are standard errors from 3 replicate isolations.