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FAQs : miRNA PCR Array & Assay

Real-time PCR and PCR Array Technical FAQ
Q: Can miScript miRNA qPCR Assays detect single nucleotide difference between miRNAs?
A: Yes. With our patent pending proprietary technology, miScript miRNA qPCR Arrays and Assays can distinguish miRNAs that differ by only one single nucleotide.

Q: Can miScript miRNA qPCR Assays be used for pre-miRNA detection?
A: No. miScript miRNA PCR Arrays and miScript Primer Assays are designed to measure exclusively the expression of mature miRNA, not pre-miRNA. To measure miRNA precursors use miScript Precursor Assays available through the QIAGEN GeneGlobe assay search portal.

Q: Do I need the sequence specific miRNA primers in the RT step of the miRNA qPCR Assays?
A: No. Because the miScript miRNA qPCR assay technology uses universal reverse transcription in the RT step, sequence specific miRNA primers are not needed in this step. Therefore, cDNA obtained from universal RT can be used for any miScript miRNA expression analysis qPCR assay or PCR array.

Q: Can I use total RNA for the miRNA PCR Arrays or Assays?
A: Yes, you can. In fact, total RNA is the recommended starting material for the miScript PCR System. We recommend using the QIAGEN miRNeasy Mini Kit (217004) to isolate total RNA for use with the miScript PCR System.

Q: Why did the real-time PCR yield Ct values < 12?
A: You probably used too much template. Try less input RNA for reverse transcription especially if the higher end of the recommended range had been used previously. Or use a smaller volume of template per PCR reaction or use diluted template, but do not use less than 1 µl of template per each 25-µl PCR array reaction. Remember to use the same volume of template in each reaction.

Q: Will pipetting error affect the miRNA qPCR Assay results?
A: The passive reference dye, ROX in the miScript PCR master mix is used by real-time PCR systems to normalize variation from well to well. Therefore, these systems tolerate volume variations caused by pipetting error and evaporation.

Q: How can I prevent the evaporation of reaction volume from the wells?
A: Be sure to carefully and completely seal the PCR plate with the optical thin-wall 8-cap strips or the optical adhesive film before placing it into your thermal cycler. Also, be sure to use a compression pad when using an optical film seal as directed by the manufacturer of your real-time PCR instrument.

Q: How reliable are the results from the miScript Primer Assays?
A: Assuming the use of good, consistent experimental technique, real-time PCR methods such as the miScript Primer Assays provide very reproducible results. To insure the reliability of your results and to reliably detect smaller fold changes in miRNA expression from the PCR Assays, the performance of replicate determinations (such as biological triplicates) is highly recommended.