The QuickExtract™ FFPE RNA Extraction Kit provides a fast, simple, and
inexpensive method for preparing RNA from formalin-fixed, paraffin-embedded
(FFPE) tissue samples for RT-PCR or real-time RT-PCR (Fig. 1). The kit requires
only heat treatment to melt the paraffin, lyse cells, decrease formalin-induced
cross-linking, and degrade compounds that may inhibit amplification. Optional
DNase reagents are included for use in some downstream applications. Following
heat treatment, the RNA sample is ready for RT-PCR.
While RNA from FFPE samples is often degraded and of poor quality, we have
demonstrated that multiple transcripts can be extracted (Fig. 2) and that full-length
coverage of the desired transcript is also possible (Fig. 3). The protocol is
ideal for high-throughput applications, with minimal hands-on time required
to process the sample. An optional protocol allows for simultaneous extraction
of both RNA and DNA from the sample.
Figure 1. Overview of the QuickExtract™ FFPE RNA
Figure 2. RT-PCR amplification of a series of different
messages present in skeletal muscle RNA. RNA was extracted from slide-mounted,
FFPE human tissue samples per the protocol and reverse transcribed with
the MMLV Reverse Transcriptase 1st Strand cDNA Synthesis Kit and random
primers. Skeletal muscle cDNA was amplified to produce short amplicons from
a number of different messages. The products were separated on a 3% agarose
gel and were visualized with SYBR® Gold. Lane M, 100-bp DNA ladder;
lane 1, a 116-bp region of RYR1; lane 2, a 162-bp region of ACTA; lane 3,
a 166-bp region of RSP18; lane 4, a 166-bp region of ENO3; lane 5, a 226-bp
region of GAPDH; lane 6, a 232-bp region of OAZ1; lane 7, a 307-bp region
of ACTB; lane 8, a 308-bp region of TNF.
Figure 3. PCR amplification of five regions of the 15.4-kb
ryanodine receptor 1 (RYR1) cDNA. RNA was extracted as per the protocol
from FFPE slide-mounted human skeletal muscle tissue samples and reverse-transcribed
with the MMLV Reverse Transcriptase 1st Strand cDNA Synthesis Kit and random
primers. The RYR1 PCR products were separated on a 3% agarose gel and were
visualized with SYBR® Gold. Lane M, 100-bp DNA ladder; RYR1 regions
amplified: lane 1, 123-228; lane 2, 2,866-2,987; lane 3, 9,132-9,279; lane
4, 11,842-11,979; lane 5, 14,935-15,097.