: miRNA PCR Array & Assay
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
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
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 <
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
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
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.