SenseAMP RNA Amplification Kit From Genisphere

Pathologists largely depend on samples archived as formalin-fixed paraffin embedded (FFPE) tissues for their research. Utilization of these samples for molecular biology experiments is greatly limited due to the degradation of nucleic acids to varying extents during fixation. There are several kits in the market for extraction of DNA/RNA from FFPE tissues. However, the yield of RNA from these samples is very low and is partially or fully degraded and not usable for downstream real-time PCR or microarray experiments. We were able to overcome this major limitation by using the SenseAMP RNA Amplification Kit from Genisphere.

This kit is the method of choice for RNA amplification from intact and/or degraded samples. The kit is optimized to amplify RNA by >1000 fold with 1 round of in vitro transcription. It requires input total RNA as little as 25 ng to a maximum of 2 µg of intact RNA. This is extremely useful as some tissues like skin biopsies are very small and give very low yields of RNA. With this range of input RNA, the same method can be adopted for samples giving both higher and lower yields of total RNA. We generally start our reaction with ~500 ng total RNA and end up getting between 10 and 20 µg of amplified RNA (aRNA) depending on the intactness of starting total RNA. This broad range is because the final yield depends on different parameters like age of the paraffin block, method and extent of fixation, etc. The older the block, the lesser the yield of aRNA will be for the same amount of starting total RNA. We used the aRNA for hybridization on glass microarrays and were able to get fairly good results. The amplified RNA is also compatible with downstream qRT-PCR and GeneChip arrays.

The SenseAMP kit uses both random and oligo-dT primers. This is also useful in amplifying partially to fully degraded RNA samples. It is important to keep in mind that aRNA from FFPE tissues will be fragmented and hence care must be taken while designing primers for qRT-PCR. The final aRNA produced is in the sense orientation which is identical to the original mRNA and hence can be labeled using conventional methods. Because this kit does not employ second-strand synthesis, they have a faster and easier protocol. One other advantage of using SenseAMP is that it produces nearly full-length transcripts, avoiding the 3’ bias of other amplification methods. SenseAMP amplification begins by synthesizing cDNA from the 3’ end of the message (dT priming). The T7 amplification reaction that follows initiates at the 3’ end of the cDNA (originally the 5’ end of the RNA). Therefore, both ends of the message are represented during the process. In contrast, conventional Eberwine-based amplification methods prime and amplify from the 3’ end of the message, typically resulting in a 3’ bias.

In general, I will say my experience with the SenseAMP RNA Amplification Kit from Genisphere has been satisfying so far.