Formalin-fixed paraffin-embedded (FFPE) tissue samples or formaldehyde fixed cell samples are a potentially valuable resource of expression information for medical research, but are under-utilized due to degradation and modification of RNA. The fixation processes preserve the tissues and cells for later analysis (e.g. using laser capture microscopy (LCM)); however, these fixation steps can lead to degradation and chemical modification of the RNA, including extensive crosslinking between biomolecules, thus hindering analysis.
The ParadisePlus Reagent System, and the ParadisePlus 2 Round Kit from MDS Analytical Technologies comes with reagents for processing 6 samples. The kit includes solvents and stain for visualizing tissues or cells on a slide, all reagents for extraction and isolation of RNA as well as the necessary compounds for a two-round amplification reaction. Depending on the application, the kit can be combined with different labeling kits, including biotin, Cy3, Cy5 and amino-allyl labeling. The kit is delivered in different boxes dependent on applications and storage conditions which makes the handling very convenient. I also liked the included plastic slide jars for easy staining of the sample slides before starting with LCM.
This kit gets used for staining, and therefore, visualizing paraformaldehyde fixed cells before performing LCM. For isolation of the RNA and all the amplification steps, I followed the very detailed protocol which is divided into sections describing the steps point-by-point in a user-friendly fashion. In addition, the reagents are color-coded for easier handling.
One of the advantages of this kit is its optimization for the use of all reactions in conjunction with LCM, especially if you are using the microdissection systems offered by the same company (Arcturus Veritas). Another important feature is the ability to use very small amounts of starting material (as little as 5 ng RNA) while still being able to perform gene expression profiling after using the amplification steps.
The beauty of the kit is its very detailed protocol as well as the color code for the reagents. The whole experiment from staining to the last clean-up step of the amplified RNA takes around three days. After LCM, the samples are digested using the included proteinase K. The duration of the digest depends on the degree of crosslinking; the amount of proteinase K depends on the size of cap which was used for the LCM processing (i.e. macro, or HS caps from MDS). The RNA is isolated using a spin column approach, which worked very well in my experience. The RNA amplification is a two step, two round process, including 1st and 2nd strand synthesis followed by an in vitro transcription reaction. The kit is optimized for using Superscript III (Invitrogen) for the 1st strand reaction. This enzyme is not included which I think is very inconvenient since purchasing the enzyme results in also getting buffers you don’t need with this kit. Regardless of all the positives about this kit, the use of T7 oligo(dT) primer instead of random priming is a major drawback. RNA isolated from fixed samples by LCM is always fragmented to a certain degree. Therefore, using oligo(dT) primer could potentially result in a 3’ bias. This is something to keep in mind when choosing the downstream application.
In my hands, the amplification worked very well. Starting with 200 ng RNA, I got 13 µg of amplified RNA.
Altogether, I think the kit performs very well, and is easy to use thanks to a detailed protocol and a nice color code.
Research Assistant Professor
Department of Surgery, Division of Vascular Surgery
University of Utah