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For best results load 0.5–1.5 µg of total RNA in a 25 µL volume per well (60 ng/µl).
RNA isolation methods validated for use with the Multiplex miRNA Cellular Assay are:
For best results isolate total RNA from cells or tissues using the TRIzol® standard protocol 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. Be aware that some degree of expertise is necessary for optimal sample recovery, as carry-over of phenol may inhibit accurate quantification of the resulting samples.
Commercially available RNA isolation kits
For best results follow the manufacturer's protocols for isolation from cells, tissue and FFPE tissues.
*Lower yield may occur using Norgen kits, but this varies on experience.
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 Multiplex Circulating miRNA Assay.
Isolated total RNA from cells and tissues
For best results, use any of the methods recommended for the Multiplex Cellular 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 Multiplex Circulating miRNA Assay can be performed even on samples that are not adequately quantified.
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