Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 7)

SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual | 1
Protocol
This protocol is intended for use with SYBR ® Green-based assays on all real-time PCR systems
using a broad range of cycling conditions, template and primer input concentrations, and fast or
standard run times.
Sample Preparation Considerations
RNA Samples
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Isolate RNA using the appropriate method for the given sample type (Aurum ™ total RNA mini
kit for cell lines, Aurum total RNA fatty and fibrous tissue kit for tissue samples)
■ ■
Compare the expected yield to the actual yield to ensure the isolation method yielded the
appropriate RNA concentrations (5–30 pg per cell, 0.1–4 µg per mg of tissue). When the yield
is less than expected, this may lead to suboptimal qPCR data results, due to less than ideal
quality samples resulting from suboptimal sample prep workflow
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When the RNA will be used for RT-qPCR, it is recommended that you treat the sample
with DNase to remove residual contaminating DNA. DNase treatment is also a good idea
when isolating RNA from tissues that are high in DNA, as the excess DNA may affect
downstream applications
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Store the RNA in an appropriate solution
– 0.1 mM EDTA (in DEPC-treated ultrapure water)
– TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0)
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Store the RNA at –80ºC in single-use aliquots
RNA Integrity and Purity
■ ■
Use the Experion ™ automated electrophoresis system or the Agilent Bioanalyzer to evaluate
the integrity of the RNA sample. When using multiple samples in the comparison, ensure that
the RQI/RIN numbers are similar to ensure accurate qPCR results
■ ■
Use an agarose gel to assess RNA integrity if the above systems are not available. Apply the
same analysis concepts. High quality RNA will yield two clean peaks, 18s and 28s. Degraded
RNA will appear as a smear on the gel
■ ■
To assess purity, evaluate the following spectrophotometer readings:
– A260/A280 >2.0 for pure RNA
– A260/A230 ~2.0 for pure RNA
• Lower ratios are indicative of contaminants from salts, carbohydrates, peptides, proteins,
phenols, and guanidine thiocyanate