RNA amplification using the Van Gelder and Eberwine technique [1] 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

The single most important factor in RNA amplification is the quality of the RNA used in the procedure. RNA quality is the sum of both integrity and purity. Intact RNA that contains trace contaminants is reverse transcribed poorly and subsequently yields less aRNA than pure samples. Conversely, RNA with 28S:18S rRNA ratios from 1-2, although lower than what is considered highly intact RNA (1.8-2), often yields high quality aRNA, as long as it is free of trace contaminants. Therefore, it is important to use a purification method that yields RNA free of contaminants.

Quantitate Total RNA

For consistency, be sure to accurately measure the concentration of the RNA to be amplified. Using those results, determine the volume of each sample needed to ensure that each reaction contains the same mass amount of total RNA. Differences in the amount of input RNA sample will result in inconsistent aRNA yields and will appear to affect amplification efficiency.

Start Small and Use Controls

When attempting RNA amplification for the first time, set up 2-3 tubes of a control RNA sample that has been qualified for microarray use, so that you can focus on your technique as you complete each step in the protocol. Assess the size and yield of replicate aRNA samples for consistency before amplifying important samples. It is not unusual to see both size and yield of aRNA improve as a researcher gains experience amplifying RNA. The best way to improve the quality of your amplification product is to pay careful attention to the amplification protocol and allow yourself time to develop competency.

More Input RNA Is Not Always Better

Exceeding the recommended amount of input RNA will result in decreased yield and size of aRNA products; typically 100-2000 ng of total RNA or 10-100 ng poly(A) RNA is recommended.

Incubation Recommendations

Incubation times: Keep incubation times as close as possible to the recommendations in the protocol. The in vitro transcription (IVT) incubation time can be as short as 4-8 hours to as long as overnight. Using the longer overnight (14 hours) incubation may increase yield (depending on the amount of input RNA used in the reaction), but it may also increase the risk of generating shorter amplification products, particularly if you exceed the recommended RNA input. IVT incubation times longer than 14 hours can adversely affect aRNA size and should be avoided. Use the same IVT incubation conditions for all samples in a study to minimize variability.

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

Many amplification protocols have a step following cDNA synthesis where the volume of the cDNA or labeled nucleotide is reduced using a vacuum centrifuge concentrator in preparation for the in vitro transcription step. The progress of drying should be watched closely because incompletely resuspending reagents that have been dried to completion can impact the efficiency of in vitro transcription, resulting in inconsistent yields. To avoid this, carefully monitor sample volume during vacuum concentration to prevent drying to completion.

Use Good General Lab Technique

The following is a list of common laboratory techniques that are important for the success of any procedure, but have proved to be particularly important in a multistep protocol like RNA amplification. Even if time is limited, do not cut corners with these basic techniques.

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.