For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
Read our guidelines detailing RNA purification sample preparation for your FirePlex? miRNA assay.?
For optimal results load between 0.1–5.0 ng of total RNA in a 25 μL volume per well, or add 40 μL plasma/serum to the digest step.
RNA isolation methods for different starting material are below:
Plasma or serum samples
For best results, store the sample at -80°C and limit the number of freeze/thaws that the samples undergo prior to quantification with the FirePlex miRNA Assay.
Isolated total RNA from cells and tissues
For best results, use any of the methods recommended for the FirePlex miRNA Assay.
Isolated total RNA from plasma or serum
For best results, isolate total RNA from plasma or serum using the TRIzol-LS?? standard protocol as applied to 250 μL of sample, with 1 μL 15 mg/mL GlycoBlue? added prior to precipitation. After precipitation with isopropanol, wash once with 75% ethanol then dry the pellet and resuspend in 50 μL RNase-free water. We suggest that 40 μL sample-equivalents be run (i.e. 8 μL of the 50 μL + 17 μL RNase-free water = 25 μL sample for assay).
Caution: For any column-based RNA isolation methodology, please confirm that the protocol you are using does not select against small RNAs. It is important to carefully follow the manufacturer's recommendations regarding ethanol concentration during wash and rinse steps to ensure that miRNAs are not excluded from the final product. Also, regardless of isolation method, it is important that the RNA be resuspended in RNase-free water or 1X TE to keep salt to a minimum.
Quantification of RNA from samples such as 250 μL plasma/serum will be impossible using absorbance-based technologies including Nanodrop and may be unreliable from more sensitive technologies such as Promega Quantus. Reliable profiling of miRNAs using the FirePlex miRNA Assay can be performed even on samples that are not adequately quantified.
FirePlex? is the new name for our Firefly assays.