mRNA Amplification from FFPE Tissue
 
Figure 2A. Gel electrophoresis of intact RNA and RNA obtained from mouse liver FFPE tissue. The positions of the 18s and 28s ribosomal bands are indicated.
Figure 2B. Size distribution of amplified RNA/cDNA generated from the FFPE tissue shown in Figure 2A by either the T7 IVT method or the Full Spectrum method. 100bp size markers (M).
To test the ability of the Full Spectrum Kit to amplify RNA derived from FFPE tissues, we isolated RNA from two 20mm sections of mouse liver. As can be see in Figure 2A, the RNA from this tissue is substantially degraded. Following amplification, we ran samples of the cDNA product on a gel. As can be seen in Figure 2B, the results show a range of cDNA products which are all relatively small in size. We also amplified a similar amount of RNA using T7-based in vitro transcription and, as anticipated, the amplified products look similar. However, since the Full Spectrum method does not rely on poly-A priming like the T7 approach does, the Universal Primer should amplify all regions of the transcripts relatively equally.
Thus, 5’-portions of the transcripts should be present in the Full Spectrum product. To test this, we amplified both samples with the transferrin receptor primers whose locations are shown in Figure 2C, along with b-actin control primers. The results of this analysis are shown in Figure 2D and support the conclusion that all regions of the transcripts are amplified relatively equally using the SBI Full Spectrum RNA Amplification Kit.