The identification of global gene expression patterns and specific gene expression levels are common goals of researchers. As evidence of this, microarray analysis has become very popular all over the world, and confirmation of microarray results by real-time PCR is essential. Although cell line RNA expression data are valuable in understanding global gene expression patterns, analyzing gene expression patterns in tissues facilitates retrospective studies which correlate gene expression patterns with the relevant disease states.
In vivo profiles may serve as biomarkers for disease classification, staging of patients and other diagnostic or prognostic purposes. Formalin–Fixed, Paraffin-Embedded (FFPE) tissue samples are the most readily available materials for human disease studies with well-documented, clinical outcomes. But RNA extracted from FFPE blocks is often too cross-linked to effectively serve as a substrate for downstream assays. Total RNA isolated from FFPE tissue samples potentially represents an invaluable resource for studying gene expression analysis. The Optimum™ FFPE RNA Isolation Kit is a simple and rapid method for isolating total RNA from FFPE samples with high-quality results. The kit is optimized for downstream real-time PCR assays.
The tissue sections should be 10-20 microns thick. The recommended number of sections used should be determined according to the type of tissue. Like in other RNA isolation protocols, an excess amount of tissue results in poor yields of RNA. After sectioning the paraffin block, the procedure begins with a deparaffinization step. The kit recommends xylene, which is not supplied with kit, for this purpose. You apply 1 ml xylene to each sample and vortex vigorously for 10 seconds. And then leave the tubes at room temperature for 10 minutes. Then, you obtain a loose pellet of tissue by centrifuging for 5 minutes. Remove the upper xylene phase by pipette gently, and prevent the pellet from drying. If you are concerned about complete deparaffinization, you may perform a more rigorous xylene treatment at 37-55ºC for up to 30 minutes. The deparaffinization step is repeated two times. And then, wash the pellet with 100% ethanol at room temperature. After vortexing the tube, spin it for two minutes and remove the ethanol by aspiration. Repeat the washing process two times more with 90% ethanol and 70% ethanol, respectively. Then, the pellet should be dried. The RNA isolation step begins with Proteinase K treatment. The Proteinase K treatment is rather long; it takes at least 3 hours at 37ºC. You should flick the tube a few times during the incubation. Check the tissue pellet after Proteinase K incubation for complete digestion. Then, add RNA extraction buffer to each sample and vortex vigorously for 10 seconds, add ethanol to each sample and again vortex vigorously for 10 seconds. After labeling the micro filter cartridge, transfer half of the sample (235 ul) and centrifuge, then transfer the remaining sample and centrifuge for an additional minute. Next, wash the filter cartridge with wash solutions 1, 2 and 3. Then dry the filter completely by centrifuging. The next step is to elute the RNA from filter with 20 µl pre-heated elution solution. The kit supplies DNase treatment agents; I would recommend this step strongly. After obtaining the eluted RNA, add the DNase buffer and DNase I to the tube, and incubate 30 minutes at 37ºC. Then apply the DNase inactivating agent to the sample, mix by vortexing, and incubate for 2 minutes. Pellet the DNase inactivating agent by centrifuging. Then transfer the RNA to a new RNase-free tube.
The Micro Filter Cartridge allows RNA to be eluted in a concentrated form. The kit is also compatible with cells from LCM (laser capture microdissection) recovered cells. It is designed for 30 reactions. One of the best things about the procedure is that it does not use phenol:chloroform extraction. This makes the kit ideal for processing many samples. The procedure is not complex, and it does not include many steps, so sample loss is minimized.
From our experiences, we can say that the real-time PCR results are satisfactory when using RNA purified with this kit. We have used this kit to isolate RNA from formalin-fixed, paraffin-embedded malignant and normal liver tissues. We use Fermentas’ cDNA synthesis kit to synthesize cDNA before PCR. We performed real-time PCR with SYBR® Green. The RNA is pure and while the Optimum® FFPE Isolation Kit provides a robust protocol, there are other uncontrollable factors, such as FFPE tissue sample preparation and/or incomplete dissociation of paraffin that may influence the overall quality and yield of RNA isolated from FFPE tissue samples.