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First Strand cDNA Synthesis Kit for RT-PCR (AMV) Protocol

Product No. 11483188001

Protocol

HANDLING OF RNA

  • Storage

Short-term storage:
Resuspended RNA is best stored at -15 to -25 °C in RNase-free buffer TE buffer (10 mM Tris, 1mM EDTA) or RNase-free H2O (with 0.1 mM EDTA).
Note: It is important to use an EDTA solution known to be RNase-free for this purpose (older EDTA solutions may have microbial growth that can contaminate RNA sample with nucleases). RNA is generally stable at -80° C for up to a year without degradation.

Long-term storage:
RNA samples can also be stored up to -80 °C as ethanol precipitates.
Note: RNA is most stable in an NH4OAc/ethanol precipitation mixture at -80 °C (although, if necessary, RNA can also be resuspended in water or buffer for storage at -80 °C).

Alternative storage of purified RNA:
Store RNA as a pellet in 70% ethanol or by adding 2 volumes of 100% ethanol to the resuspended RNA. Recover the RNA by adding sodium acetate and centrifuging.

Generally, it is recommended that RNA samples be aliquoted into several tubes. This will both prevent damage to the RNA due to successive freeze-thawing, and help to prevent inadvertent RNase contamination.

  • Recovering and Dissolving RNA

After centrifugation, RNA should be dried briefly at +37 °C, or in a vacuum oven if available.
Note: Avoid completely drying the RNA pellet, because this makes RNA difficult to resuspend. When working with RNA, place all samples on ice. For reasons mentioned above, RNA is very susceptible to degradation at room temperature. Dissolve RNA by adding RNase-free buffer TE buffer (10 mM Tris, 1mM EDTA) or RNase-free H2O (with 0.1 mM EDTA); then incubate the tube on ice for 15 min. Gently tap or carefully vortex the tube to resuspend RNA.

  • Precipitation of RNA for downstream applications

When more concentrated purified RNA is required for downstream applications, an ammonium acetate (NH4OAc) precipitation (0.1 volumes of 5 M NH4OAc, and 2-2.5 volumes 100% ethanol, at -20 °C for >25 min) results in excellent recovery of RNA. For quantitative recovery of low concentrations of RNA (ng/ml), an inert coprecipitant, such as glycogen, yeast RNA, or linear acrylamide, should be used.

Materials

     
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