RNAstorm™ and DNAstorm™ FFPE extraction kits
Contamination from genomic DNA is a big concern because it can interfere with downstream applications. The RNAstorm™ kit includes an optimized DNase digestion step that removes contaminating genomic DNA without significantly affecting RNA yield. While this step is optional, it is highly recommended.
The most significant variable that affects the total amount of RNA or 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). Assuming a reasonable sample quality, you can expect to obtain more than 1 ug using the RNAstorm™ or DNAstorm™ kits.
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 that provide at least 1 µg of RNA should be used.
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 that is not embedded in paraffin can be used with these kits. In this case, we recommend mechanically grinding an amount of tissue equivalent to the recommended number of sections. If you are isolating RNA, we also have an RNAstorm™ kit for isolating RNA from fresh or frozen cells or tissue samples.
The RNAstorm™ FFPE and DNAstorm™ FFPE extraction kits include 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 mixed vigorously. To avoid this issue, we recommend not vortexing the sample when the Deparaffinization Reagent and CAT5 Lysis Buffer are in contact. Instead, we recommend pipette mixing when necessary in the presence of both these reagents (e.g., when protease is added). The white cloudy layer can be removed by centrifuging the sample 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 10kb) in better-quality samples.
Yes. Good quality libraries can be obtained, providing that the DNA is sufficiently high quality.
Contamination from RNA is eliminated by performing an optimized RNase digestion step immediately following the lysis step.
The maximum capacity of the spin columns in the kit is similar to a standard miniprep column, about 20 ug of DNA. However, the expected yield from FFPE extraction is much lower, it is very rare to get more than 1-2 ug of DNA per prep.
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:
- Fragment size distribution: a 5 µg sample (as measured by Qubit) can be useless for RNA-Seq if it consists of fragments < 200 nt.
- Chemical modification: for RNA obtained from formalin-fixed samples, various chemical adducts and crosslinks, including base modifications, base-base crosslinks, and base-protein crosslinks can make nucleic acid molecules inaccessible to enzymes and therefore inactive in downstream applications.
- Contamination: cellular debris, proteins, salts, and detergents used during purification can bias downstream assays. For example, UV/Vis-based methods such as Nanodrop are particularly susceptible to contaminants that absorb in the 200-280 nm range.
- Fluorescence-based methods such as Qubit are liable to significant error. When working with low concentrations of DNA or RNA, dye-based detection may not be linear. One must also be mindful of contamination by genomic DNA in an RNA sample because the dyes used for fluorescence quantitation are not entirely specific for FFPE-derived DNA or RNA.
- Quantitative PCR is the preferred method for quantitation of heavily damaged and modified nucleic acids.
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.
- Avoid methods based on organic solvents (Trizol)
- Avoid harsh chaotropic salts (i.e. guanidinium)
- Avoid detergents that impact downstream quantitation by UV and/or Qubit (e.g. Triton X-100)
- Do not rely on RIN to quantitate the integrity of an FFPE-derived sample, use DV200 instead.
- Use a kit or method that removes chemical modifications from formalin. Do not raise the temperature to 80˚C or above. Even short times at this temperature will significantly lower integrity.
- Be wary of Qubit and Nanodrop concentrations because of the possibility of contamination by organic molecules or DNA.
- Use qPCR to quantitate your RNA, and always look carefully at melt curves to determine whether nonspecific amplification may have occurred.
PCR inhibition is often observed when high amounts of FFPE-extracted template DNA are used. The inhibition is not usually 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.
Yes, the RNAStorm™ and DNAStorm™ FFPE kits may be used sequentially. The steps below will allow the protocol to be adapted to extract both RNA and DNA from one sample. You can also download this information in an app note.
Begin by extracting the sample according to the RNAstorm™ kit protocol with the following modifications:
- Perform step 3 (normally a 2 hour incubation) for only 30 minutes at 72˚C. See note below regarding possible optimization of this step.
- Perform steps 4 and 5 of the RNAstorm™ protocol as directed, but do not discard the pellet (which contains the DNA) in step 5.
- Transfer the supernatant (which contains the RNA) to a new tube as instructed in step 6.
- Continue to incubate the supernatant for another 1.5 hours at 72˚C (2 hours total including the initial 30 minutes), then proceed with step 7 of the RNAstorm™ protocol (add Binding Buffer) and all remaining steps as instructed.
- Use the pellet from step 2, which contains DNA, as input for step A5 (or B8, depending on deparaffinization choice) of the DNAstorm™ kit manual.
- Continue with step A5 (or B8) of the DNAstorm™ protocol by adding 200 µL of CAT5™ Buffer to the pellet, then continue as instructed by the DNAstorm™ protocol.
Note: the initial incubation period can be adjusted depending on relative DNA and RNA yields. If the RNA yield is high but the DNA yield is low, reduce the incubation time in step 3 (no less than 15 mins). If the DNA yield is good but the RNA yield is low, increase the incubation time in step 3 (no more than 2 hours).