Ten Ways To Improve Transcription From CDNA Templates For RNA Amplification (aRNA)

Ten Ways to Improve Transcription From cDNA Templates for aRNA Amplification

10. Begin with highest quality RNA samples.
    Good RNA amplification procedures begin with intact RNA samples that are free of contaminating nucleases, salt and genomic DNA. Any contaminant that inhibits reverse transcription will reduce the yield of cDNA and subsequently reduce the final yield of amplified antisense RNA (aRNA).
    
    
9. PAGE purify the first-strand T7 oligo(dT) primer.
   These primers are typically 40 bases or longer. Truncated oligonucleotides will lack the full promoter sequence and produce non-productive cDNA templates. Ambion provides gel-purified, tested T7-Oligo(dT) Promoter Primers so that synthesis is not necessary.
   
8. Optimize the first-strand T7 oligo(dT) primer concentration for different starting amounts of sample RNA.
   Ambion has found that 50 ng of primer works well with 0.1-1 µg of poly(A) RNA. (Also see Baugh, L. R. et al. (2001) Nucleic Acid Research, 29(5): e29.)
   
7. Skip the use of E. coli DNA ligase during 2nd strand cDNA synthesis.
   Ambion and other researchers have observed that the yield and size distribution of amplified RNA is not compromised by the elimination of this enzyme from 2nd strand synthesis reaction protocols. (see Methods: A Companion to Methods in Enzymology. (1998) 16: 444-452 . Note, however, that the optimal concentration may vary with sample.
   
6. Remove rRNA before in vitro transcription.
   When amplifying mRNA from a total RNA sample, the cDNA will contain a significant amount of rRNA. This rRNA can be removed by alkaline hydrolysis (NaOH treatment followed by neutralization with Tris). Removal of the contaminating rRNA can improve amplification and eliminates rRNA carryover into downstream hybridizations.
   
5. Maximize large scale transcription reaction conditions.
   The yield and quality of the aRNA produced from a MEGAscript™ Kit is primarily determined by the quality of the cDNA template used in the reaction. However, certain steps can be taken to maximize yields during transcription. Some benefit may be gained by setting up 2X reactions (40 µl). Be sure all transcription components have been well mixed and briefly centrifuged prior to setting up reactions. Transcription reactions using cDNA templates (heterogenous templates) should be incubated for at least 6 hours. Maximal yield is obtained from overnight (18 hr) incubation. The most reproducible method for incubation is in a thermocycler with a heated lid. Some increase in yield is also observed with 1-2 µl additional enzyme.
   
4. Use DNA-free™ to eliminate DNA.
   After in vitro transcription use Ambion's DNA-free™ to destroy the cDNA template and T7 oligo(dT) primer. Perform DNase I digestion, then inactivate DNase using the DNase Inactivation Reagent (included). While the DNase I supplied in the MEGAscript Kit is sufficient for DNA digestion, DNA-free provides a reagent for complete removal of the DNase I enzyme in addition to those for DNase treatment.
   
3. Purify the amplified RNA.
   The MEGAclear Kitgives excellent recovery and removes unincorporated NTPs, proteins and DNA from transcription reactions. The majority of the RNA products below 100-200 bases will be effectively removed, thus, minimizing endogenous priming.
   
   
2. Check amplified RNA quality.
   To assess amplification efficiency, run 1-2 µg (as determined by A₂₆₀ or RiboGreen® (Molecular Probes)) of your RNA product on a denaturing agarose gel stained with ethidium bromide. This gel should show a smear of RNA from 500 to 4000 nucleotides (nt). A preponderance of small molecular weight products (~100 nt) could indicate that the amplification was not optimal or the starting mRNA was degraded. Ambion uses the Agilent 2100 bioanalyzer for routine analysis of aRNA, mRNA and total RNA. This instrument can accurately measure amplified RNA amounts as low as 25 ng.
   
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   Now a Complete Kit for RNA Amplification
   Ambion's new MessageAmp™ aRNA Kit contains reagents optimized for each step of the aRNA procedure and includes the patented MEGAscript technology for high-yield in vitro transcription. While yields will vary depending on the RNA source and quality, a typical reaction will yield as much as 10 µg of aRNA starting from 1 µg total RNA (this represents 1000X amplification of the mRNA present in 1 µg of total RNA). Smaller amounts of total RNA (10 ng) can be used with 2 cycles of amplification to generate 10 µg or more of aRNA.