Tips for Successful RNA Amplification
|RNA amplification using the Van Gelder and Eberwine technique  is a multistep protocol comprised of several enzymatic and clean-up steps. Organization, technique, timing, and equipment are all critical to producing high quality amplified antisense RNA (aRNA). Since aRNA quality is key to obtaining reproducible array data, it is imperative that the techniques used for amplification are carried out with the utmost care. Here, we list our recommendations for performing consistent and reproducible RNA amplifications for microarray analysis.|
Use High Quality RNA
Quantitate Total RNA
Start Small and Use Controls
More Input RNA Is Not Always Better
Incubation Temperatures: Another critical factor is variable or inaccurate incubation temperatures, which can limit both cDNA and aRNA synthesis reactions. The incubator type is also important because condensation will change the composition of the reaction mixture and can reduce both size and yield of resulting aRNA. For the most consistent results, we recommend conducting 70°C denaturation and 16°C second strand synthesis reactions in a thermal cycler. Thermal cyclers are ideal for high temperature denaturation because the reaction solutions reach the target temperature rapidly. For the critical second strand synthesis reaction, a thermal cycler is by far the best way to maintain a uniform 16°C reaction temperature for 2 hours. Newer thermal cyclers have heated lids that mimic the temperature of the block; with these machines, we do recommend using the heated lid for these incubations. If, on the other hand, you are using a thermal cycler with a single-temperature heated lid (typically ~95°C), use it with the heat turned off. If your thermal cycler does not have that option, incubate reactions without the lid, otherwise the heat from the lid will raise the temperature of the reaction--this would be especially detrimental for the second strand synthesis reaction. Check with the manufacturer of your thermal cycler to find out how your machine functions. For all other incubations in amplification procedures, we recommend using a hybridization oven or a constant temperature incubator because in these devices the heat envelops the tubes, minimizing condensation. To maintain consistency, never place reactions in a thermal cycler or incubator until the temperature has stabilized.
Don't Overdry cDNA or Nucleotides
Use Good General Lab Technique
Make master mixes
Master mixes should be prepared when processing 2 or more samples simultaneously to reduce the number of pipetting steps and the potential for pipetting error. Always include ~5% overage of all reagents in master mixes to cover pipetting error.
Thaw all reagents properly
All frozen reagents should be thawed completely, mixed thoroughly, centrifuged briefly, and placed on ice as necessary. However, allow IVT components to equilibrate to room temperature before setting up your reactions because spermidine in the reaction buffer may cause cDNA to precipitate at lower temperatures. To ensure optimal performance, thaw components at room temperature, and avoid higher temperatures.
Be gentle with enzymes
Never vortex enzymes. Mix by gently flicking the side of the tube to avoid inactivating the enzyme.
The success of your microarray analysis depends on the quality of the aRNA used in the hybridization. Following these tips and reminders for amplification can greatly increase the likelihood of obtaining good quality aRNA and reproducible microarray data.