RNA Handling When Using the First Strand cDNA Synthesis Kit for RT-PCR (AMV)

The First Strand cDNA Synthesis Kit (Product No. 11483188001) is used for the synthesis of the first strand cDNA as the starting reaction for two-step RT PCR.The kit includes Reverse Transcriptase AMV, two different primers, PCR nucleotide mix, and Control Neo pa RNA. The PCR products that are generated by RT-PCR can be cloned using standard procedures.

The amplification of RNA requires the conversion of the RNA substrate into DNA, which is achieved using the reverse transcriptase AMV RT (avian myeloblastis virus reverse transcriptase). The resulting cDNA can be used as a template for a standard PCR. AMV RT synthesizes the new cDNA strand at site(s) determined by the type of the primer used:

• the 3′-end of the poly(A) mRNA when Oligo-p(dT)₁₅is used as a primer
• nonspecific points along the mRNA template when using the random primer p(dN)₆
• site determined by a sequence-specific primer

Handling of RNA

Short-term storage:
Resuspended RNA is best stored at -15 to -25 °C in RNase-free buffer TE buffer (10 mM Tris, 1 mM EDTA) or RNase-free H₂O (with 0.1 mM EDTA).
Note: It is important to use an EDTA solution known to be RNase-free (EDTA solutions stored for long periods may have microbial growth that can contaminate RNA sample with nucleases). RNA is generally stable at -80° C for up to a year.

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 two 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 caused by repeated 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.
Note: Avoid completely drying the RNA pellet, which makes RNA difficult to resuspend. When working with RNA, place all samples on ice. For reasons mentioned above, RNA is susceptible to degradation at room temperature. Dissolve RNA by adding RNase-free TE buffer (10 mM Tris, 1 mM 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.