Using the FirePlex miRNA Assay
For the best results 0.1–0.5 ng of total RNA should be loaded in a volume of 25 µL per sample well, or to the digest step add 40 µL of plasma or serum.
Methods for RNA Isolation
Total RNA isolated from cells and tissues: For optimal results, any of the methods given for the FirePlex miRNA assay can be used.
Total RNA isolated from plasma or serum: For optimal results, total RNA should be isolated using the TRIzol-LS® standard protocol with 250 µL of sample and the addition of 1 µL 15 mg/mL GlycoBlue™ before precipitation. Following isopropanol-induced precipitation the pellet should be washed with 75% ethanol then, following drying, the sample should be resuspended in 50 µL water free from RNase. It is suggested that sample-equivalents of 40 µL are ran, i.e. 8 µL of the 50 µL + 17 µL RNase-free water = 25 µL sample for assay.
Plasma or serum samples: For optimal results, the sample should be stored frozen at -80 °C with the number of freeze/thaws the sample undergoes as low as possible. Methods given for the FirePlex miRNA assay can be used.
When running any RNA isolations that use a column, care must be taken that the method does not have negative bias towards small RNAs. Recommendations regarding ethanol washing and rinsing procedures should be adhered to so that miRNAs are not excluded from the final pellet.
For all isolation methods, it is important that the RNA is resuspended in water that is RNase free, or 1X TE, to keep salt levels low.
Quantifying the RNA in samples like 250 µL of serum or plasma cannot be achieved using absorbance technologies such as Nanodrop, and may also be unreliable using more sensitive systems such as Promega Quantus.
Accurate miRNA profiling can be carried out on samples, regardless of if they have been quantified, using the FirePlex miRNA Assay.
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