Overview
Both mammal and plant tissues can be “imprinted” on the cards simply by pressing the material onto the sample spotting area. Saliva, blood, mucus, cerebral spinal fluid, and any other fluid of interest can be spotted onto the cards at a volume of up to 125 microliters, depending on the type of FTA card you are using. Once the cards are dry, they can be stored at room temperature for up to several months (Whatman’s site claims years, but I am skeptical) before attempting to extract the DNA or the RNA from the sample. To extract the nucleic acids, you first need to take a punch of the card where your sample is imprinted. The Harris micropunch, though expensive, is quite popular. Others have found that sterilized scissors and forceps, rinsed with ethanol and flamed between samples, are effective. A metal hole puncher purchased from a craft store is also a possibility. Some researchers say that, while the FTA cards are convenient on the sample collection end, the downstream processing is often not as pleasant. In particular, it is not likely that you will be able to recover 100% of the nucleic acid from your sample, so optimizing your extraction protocol should aim to maximize recovery.Extracting nucleic acids from FTA cards
Whatman publishes a few protocols for extracting nucleic acids from the FTA Cards, which are available for free online, like this one for DNA or this one for RNA, although their official protocol page is not that helpful. You can also try DIY methods or commercial kits. No matter how you slice it, most protocols fall into two categories: Direct-to-PCR and Elution.- Direct-to-PCR
In the direct-to-PCR protocol, a single 1-2 mm punch is removed from the sample area with a sterilized micropunch, washed with detergent, rinsed with buffer, and then dried on a heat block according to a standard protocol. The paper punch can then be added to the PCR tube as the template and amplified as usual. When followed exactly as written, this protocol is simple and requires very few steps. Whatman sells a proprietary card-washing reagent which is quite expensive, and the literature shows mixed success using SDS as a substitute. Whatever you choose, it should be able to wash away proteins and other chemicals and denature nucleases.
- Elution of Nucleic Acid From the Card
For very concentrated samples, elution is not much more difficult than a direct-to-PCR protocol. Things like cheek swabs, blood spots, and other sample types with large quantities of DNA can be extracted using commercial kits or Whatman protocols. The company even sells a type of “FTA Elute” card specifically designed for elution protocols rather than direct-to-PCR.
However, for projects where the samples are very dilute and the entire sample area needs to be processed (instead of just a few punches), protocol optimization becomes more complicated. Consider the challenges in processing large quantities of paper:- Volume
The amount of elution buffer required to fully submerge the paper can be at odds with the size of the tube, especially if you plan on ethanol precipitating and concentrating the nucleic acid later. That requires double the sample volume of ethanol achieve, so make sure you leave enough room! Some researchers solve this by using concentrator tubes or processing the paper directly in kits that employ the bind-wash-elute column method, but that can be expensive.
- Number of steps
It also helps to reduce the number of steps, including tube transfers, to minimize loss of product. Many kits with built-in card protocols allow you to put the paper directly into the elution medium (usually a buffer, a detergent, and proteinase K) rather than washing and drying it first. Our lab prefers kits that allow elution in very small volumes for samples of low concentration. Incubating the tubes on ice and avoiding agitating the tubes during the wash steps can help reduce product loss.
- Handling
In non-column protocols, sometimes the vortexing or shaking necessary to elute the nucleic acid can also start to break down the paper, leaving behind paper fuzz in the tube. The paper fuzz usually does not end up in the final eluate in kits with micro-column methods, but it can be frustrating when attempting to purify nucleic acid by ethanol precipitation after using a non-kit elution protocol. Some paper fuzz is usually left behind, even if you try to spin it out and transfer the supernatant to a new tube, not to mention that transferring to a new tube introduces an opportunity for product loss. Leaving the fuzz behind tends not to interfere with PCR reactions, but it does make the ethanol wash take longer to evaporate from the tube, increasing the risk that you will either contaminate your samples or incompletely dry them.