Tip 1: Coprecipitants Improve RNA Recovery Without Affecting Amplification
A detailed study by Q. Tian Wang et al (1) addresses the effect that coprecipitants have on linear amplification of RNA. Coprecipitants are often used during RNA isolation to increase RNA recovery during precipitation. This analysis was carried out using 10 ng of yeast total RNA precipitated in the presence of increasing amounts of linear acrylamide (5 to 25 ng) or using a combination of linear acrylamide (5 ng) and increasing amounts of yeast tRNA (50-500 ng) as carriers. The precipitated RNA was then converted into cDNA and amplified by linear amplification. The use of a combination of linear acrylamide and tRNA improved the total RNA recovery. Inhibitory effects of tRNA on cDNA synthesis were not observed in this study, and aRNA product sizes and yields obtained after amplification were indistinguishable from products obtained in the absence of carriers. Experiments in Ambion's own laboratories corroborate this result. Analysis of the aRNA probes prepared from RNA samples containing 0, 50, and 250 ng of yeast tRNA carrier on an Affymetrix GeneChip® system yielded average "r" values of 0.991 0.994 and 0.995, respectively. Thus the use of yeast tRNA supplemented with linear polyacrylamide allowed efficient cDNA synthesis and amplification of aRNA from nanogram quantities of starting total RNA.
Ambion offers a variety of high quality coprecipitants. These include Glycogen, GlycoBlue™ (glycogen derivatized with a blue dye to increase pellet visibility), sheared Yeast Total RNA, Yeast tRNA, and Linear Acrylamide. Each coprecipitant is subjected to stringent quality control tests to ensure that it is free of RNases as well as other unwanted contaminants.
Tip 2: Double Stranded cDNA Clean-up
Different linear amplification protocols call for different double stranded cDNA purification methods prior to amplification. This step is designed to purify the cDNA template from free nucleotides, oligo(dT)-T7 primers, and salts. A recent publication by Zhao et al (2) found that gel filtration based purification of double stranded cDNA followed by phenol chloroform extraction resulted in decreased yields of aRNA due to loss of the double stranded cDNA. At Ambion, we use a column binding and elution method that allows elution of the double stranded cDNA in small volumes. In addition to avoiding the need to phenol extract, this eliminates extra vacuum drying or precipitation steps to concentrate the cDNA before in vitro transcription and results in up to 2X higher aRNA yields compared to phenol based purification (Figure 1). The column binding method is available as the DNAclear™ Kit
for the purification of cDNA templates for subsequent aRNA amplification and is included in Ambion's MessageAmp Kits
Figure 1. Comparison of Two cDNA Purification Methods for Subsequent aRNA Amplification.
Two identical rat thymus total RNA (2 µg) samples were amplified using the MessageAmp™ aRNA Amplification Kit. During the amplification protocol, the double stranded cDNA from one of the samples was purified using phenol:chloroform: isoamyl alcohol (pH 7.9) whereas cDNA from the other sample was subjected to Ambion's DNAclear™ (column based) clean-up protocol.