Critical Parameters for Successful RNA Amplification

We recommend using a combination of a homogenization/organic extraction reagent with traditional glass fiber filter purification (Ambion's RiboPure™ RNA Isolation Kits combine these two isolation methods) to yield exceptionally pure RNA that is free of residual proteins and lipids. The type of tissue and its developmental state and health will determine the amount of mRNA recovered. mRNA makes up about 2% of a total RNA sample. Some tissues like brain, testes, or embryos may have up to 4% mRNA while many tissues may contain only 1% or less. The method of extraction can also affect the representation of RNA species recovered, including mRNA.

RNA samples should be free of contaminating proteins, DNA, and other cellular material as well as phenol, ethanol, and salts associated with RNA isolation procedures. Impurities can lower the efficiency of reverse transcription resulting in reduced amplification. RNA purity can be estimated from the ratio of absorbance readings at 260 and 280 nm. The A ₂₆₀:A ₂₈₀ value should fall in the range of 1.7-2.1. This can be accomplished by purification protocols that bind the RNA sample to glass fibers or beads.

Typically 100-2000 ng of total RNA, or 10-100 ng of poly(A) selected RNA should be used as starting template for the aRNA amplification procedure. The maximum recommended amount of RNA for this procedure is 5 µg of total RNA or 1 µg of poly(A) RNA.

The integrity of the RNA sample, or the proportion of mRNA that is full-length, is the most important component of RNA quality. Reverse transcribing partially degraded mRNAs will typically generate relatively short cDNAs that lack upstream sequences. Final yields of aRNA amplified from such cDNA will be decreased if the Poly(A) tail has been degraded. There are currently no quantitative methods for measuring the amount of full-length mRNA in a sample. However several procedures do exist for establishing the relative integrity of a sample. The most frequently used procedure for estimating RNA integrity is fractionating 2-4 µg of a total RNA sample on a denaturing agarose gel. Ethidium bromide staining of the RNA reveals the 18S and 28S ribosomal RNA (rRNA) bands. If these bands are discrete (i.e. there is no significant smearing below each band) and the 28S rRNA band is approximately twice as intense as the 18S rRNA band, then the mRNA in the sample is of good quality. The primary drawback to this method is that microgram amounts of RNA must be sacrificed. A method that requires only nanogram quantities of RNA is using the Agilent 2100 bioanalyzer and RNA6000 Nano LabChip Kit. Like a denaturing agarose gel, the bioanalyzer fractionates RNA molecules according to size, and the amounts of 18S and 28S rRNA are automatically calculated. Theoretically, intact RNA will have a 28S:18S rRNA ratio of 1.7-2.0. However, this ratio is rarely obtained with tissue samples and indeed varies from source to source.

Several different methods can be used to label RNA for array analysis. Cy™ dyes, other fluorophores, or biotin can be incorporated: 1) during the in vitro transcription step of aRNA synthesis, 2) during a subsequent reverse transcription reaction or, 3) after aRNA synthesis in a post-synthesis coupling reaction. Increasingly, amino allyl incorporation combined with a dye coupling reaction is used for the generation of probes for array analysis. This strategy offers several advantages over the direct incorporation of labeled NTPs. Direct incorporation of fluorescently labeled NTPs is inefficient, resulting in lower yields and lower specific activity aRNA. In addition, incorporation efficiency varies for different labels (e.g. Cy3 NTP vs. Cy5 NTP). Because many labeled NTPs are incorporated inefficiently, the cost of producing labeled aRNA can be very high. These problems can be avoided by incorporating an amino allyl UTP into the aRNA and subsequently coupling its reactive amino group to an NHS ester label (e.g. biotin, Cy dye). Unlike dye coupled UTPs, amino allyl modified UTPs are incorporated almost as efficiently as unmodified NTPs and are much less expensive than the dye coupled NTPs. A variety of inexpensive NHS ester dyes and other nonisotopic labels are available from Amersham Biosciences, Pierce Biotechnology, and Molecular Probes.

To assess amplification efficiency, run 1-2 µg of your RNA product on a denaturing agarose gel stained with ethidium bromide. This gel should show a range of aRNA from 500-4000 nucleotides (nt) with a modal size of approximately 1800 nt. Alternatively, the Agilent 2100 bioanalyzer can also be used. A preponderance of small molecular weight products (~100 nt) may indicate that amplification was not optimal or that the starting mRNA was degraded.

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