Contamination from genomic DNA is a big concern because it can intefere with downstream applications. The RNAstorm™ kit includes an optimized DNase digestion step which removes contaminating genomic DNA without significantly affecting RNA yield. While this step is optional, it is highly recommended.
The biggest variable that affects the total amount of RNA obtained is the quality of the sample itself (i.e. the type and amount of tissue, and the care taken in isolation and preservation of the sample). Using the RNAstorm™ kit, and assuming at least reasonable sample quality, amounts greater than 1 µg can be obtained.
Yes. Good quality libraries can be obtained, providing that the RNA is of sufficiently high quality. For Illumina sequencing, a DV200 of at least 30% is recommended, and samples should be used that provide at least 1 µg of RNA.
Use a microtome to obtain 5-10 µm sections from FFPE samples. Sections thinner than 5 µm may be used if they can be reliably cut. Sections thicker than 10 µm are not recommended because they may not be fully digested. Also, no more than 5 sections (10 µM each) should be used for each extraction. Using too much tissue can lead to incomplete digestion and reduced yields.
Yes, tissue can be used which is not embedded in paraffin. In this case, we recommend mechanically grinding an amount of tissue equivalent to the recommended number of sections.
Yes, FFPE cores can be used. Because cores are not processed using a microtome, sample digestion tends to be more difficult and mechanical homogenization (e.g. using steel beads) is recommended if incomplete digestion is observed.
The RNAstorm™ kit includes a recommended Deparaffinization Reagent. Unlike other common methods (e.g. xylenes), the Deparaffinization Reagent is efficient, non-toxic and does not require the use of a fume hood. In our testing, the included reagent is at least as effective as xylenes at removing paraffin and allowing purification of high quality nucleic acids.
FFPE-derived RNA is much more challenging to quantitate accurately than RNA obtained from fresh samples. It is not enough to know the absolute amount of RNA that is present, but also whether the RNA will work in downstream applications, which depends on the following factors:
Although the RIN number can provide general information about the extent of sample fragmentation, it is not sensitive or predictable enough to be a useful indicator of downstream performance, especially for RNA-Seq. Very often, RIN numbers for FFPE-derived RNA will be between 2 and 3. Some of these samples will be useful for RNA-Seq, and others won’t - the RIN will not tell you, however.
A slightly better predictor of performance in RNA-Seq using Illumina sequencing is the DV200, which represents the percentage of RNA fragments longer than 200 nucleotides. The DV200 is also calculated based on Bioanalyzer data, but suffers from the same drawbacks as all Bioanalyzer-based methods, specifically high variability.
Contamination from RNA is eliminated by performing an optimized RNase digestion step immediately following the lysis step.
The biggest variable that affects the total amount of DNA obtained is the quality of the sample itself (i.e. the type and amount of tissue, and the care taken in isolation and preservation of the sample). Using the DNAstorm™ kit, and assuming at least reasonable sample quality, amounts greater than 1 µg can be obtained.
Yes. Good quality libraries can be obtained, providing that the DNA is of sufficiently high quality.
Use a microtome to obtain 5-10 µm sections from FFPE samples. Sections thinner than 5 µm may be used if they can be reliably cut. Sections thicker than 10 µm are not recommended because they may not be fully digested.
Yes, tissue can be used which is not embedded in paraffin. In this case, we recommend mechanically grinding an amount of tissue equivalent to the recommended number of sections.
Yes, FFPE cores can be used. Because cores are not processed using a microtome, sample digestion tends to be more difficult and mechanical homogenization (e.g. using steel beads) is recommended if incomplete digestion is observed.
The DNAstorm™ kit includes a recommended Deparaffinization Reagent. Unlike other common methods (e.g. xylenes), the Deparaffinization Reagent is efficient, non-toxic and does not require the use of a fume hood. In our testing, the included reagent is at least as effective as xylenes at removing paraffin and allowing purification of high quality nucleic acids.
The white cloudy layer is an emulsion between the Deparaffinization Reagent and the CAT5 Lysis Buffer, which may form when these two reagents are vortexed or given a hard mix. To avoid this issue, we recommend not vortexing the sample when the Deparaffinization Reagent and CAT5 Lysis Buffer are in contact. When mixing is necessary in the presence of both these reagents (e.g. when protease is added), we recommend pipette mixing. The white cloudy layer can be removed by giving the sample a hard spin at maximum speed (> 16,000 x g) for at least 2 minutes. The length of time will depend on the volume of the emulsion.
Due to the wide size distribution of DNA isolated from FFPE tissue samples, we recommend using pulsed-field gel electrophoresis (PFGE). Methods based on capillary electrophoresis such as the Agilent BioAnalyzer can also be used, but may not properly resolve high molecular weight fragments (greater than 10k) in better-quality samples.
PCR inhibition is often observed when high amounts of FFPE-extracted template DNA are used. The inhibition is usually not due to the presence of contaminants, but results from residual chemical modifications and damage in the DNA itself. Several simple adjustments to the PCR protocol can overcome this issue. First, the amount of template DNA should be decreased. Second, the amount of PCR polymerase should be increased by 2-4x. Third, the annealing and extension times should be extended. Fourth, the amount of dNTPs can be increased.
An in-depth discussion of this issue is found in Dietrich et al. (2013), PLoS ONE 8(10): e77771.