Applications
· Isolation of RNA from archived FFPE samples for RT-PCR-based analysis.
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.
Benefits
· RT-PCR-ready extracted RNA in 30 minutes, ideal for high-throughput applications.
· No xylene or phenol extractions.
· No columns, transfers, or sample loss.
· Optional protocol allows simultaneous extraction of RNA and DNA from the same tissue sample.
Figure 1. Overview of the QuickExtract™ FFPE RNA extraction procedure.
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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.
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Catalog No. |
Size |
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QuickExtract™ FFPE RNA Extraction Kit |
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QFR82805 |
5 ml (50 Reactions) |
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QFR82050 |
50 ml (500 Reactions) |
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Includes Extraction Solution and optional DNase Reagents. |
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