It is often useful to be able to isolate both RNA and DNA from the same biological specimen, especially when the sample is in short supply or when different manipulations are contemplated (for example, genomic PCR and RT-PCR). Some protocols accomplish this aim by isolating a total nucleic acid fraction that is then divided into two portions which are treated differentially with either DNase I (to remove DNA and recover RNA) or with RNase A (to selectively recover the DNA). Two problems with this approach are that half of the DNA and half of the RNA fractions are wasted, and that if the DNase and RNase enzymes are not extremely pure, there may be degradation of the desired nucleic acid fraction.
An alternative approach is to sequentially isolate the RNA and DNA fractions from the same sample. This can be done by adapting the protocol for RNA isolation used in Ambion's ToTALLY RNA™ Kit as outlined below. As shown in Figure 1, good yields of intact RNA that is free of DNA, and of high molecular weight DNA free of RNA, were obtained from a snap frozen breast tumor biopsy specimen. Note, this tissue has a reputation for being difficult to work with due to its high content of adipose and connective tissue, but we did not encounter any special problems when using the To-tally RNA™ Kit protocols. The protocol works very well on soft tissues (e.g. liver, spleen, and cell lines). We have found the DNA recovered by this method to be suitable for use in PCR reactions and other applications. This procedure is similar to one recently published in BioTechniques (1993) 15,22-24.
Figure 1. Approximately 500 mg of Breast Tumor Tissue Was Prepared Using the Simultaneous RNA/DNA Protocol. The RNA was suspended in 400 µl RNase-free water/0.1 mM EDTA. The DNA was suspended in 200 µl TE. The samples were run on a 1%, pH 7.8 agarose gel for 25 min. at 0.6 V/cm. Lane 1: 1 µg pUC 19/Sau 3A markers, Lane 2: 1 µl breast tumor DNA, Lane 3: 1 µl breast tumor RNA, Lane 4: 5 µl breast tumor DNA.
Hair follicles were microdissected from the tissue specimens and total RNA was isolated using the Ambion RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE. RNA concentration, quality, and purity could not be accurately determined using the NanoDrop Spectrophotometer because the RNA concentration was near the instrument’s lower limit of detection (2 ng/µL). However, there was sufficient RNA to perform quantitative real-time RT-PCR to determine if the staining process had any impact on the quality or performance of the RNA.
Total RNA was prepared from both 3 and 9 microdissected follicles from unstained, Mayer’s hematoxylin stained, or cresyl violet stained preparations, converted into cDNA and analyzed for GAPDH expression using quantitative real-time PCR (Figure 2). As expected, the CT value decreased appropriately with increasing input of follicles in unstained tissue (3-fold difference = 1.6 CTs). However, use of Mayer’s hematoxylin staining had a detrimental effect on RNA analysis as measured by the increased CT value of GAPDH expression at both 3 and 9 follicles. In contrast, staining the tissue with cresyl violet from the Ambion LCM Staining Kit did not have a negative effect on RNA amplification as compared to unstained tissue.
The data demonstrate that cresyl violet staining allows for higher contrast and more intense cellular staining compared to either 10% Mayer’s hematoxylin or unstained samples, and does not interfere with downstream RNA analysis.
The study concluded that the Ambion LCM Staining Kit could successfully stain FFPE tissue sections for LCM procedures. In combination with the Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE, the quality of the subsequently isolated RNA is comparable to that of unstained control LCM tissue.
Figure 2. Real-Time RT-PCR Data from Total RNA Isolated from Unstained or Cresyl Violet-Stained FFPE Tissues. RNA (10 µL) was reverse transcribed with M-MLV reverse transcriptase and random primers. cDNA was used in real-time PCR (ABI PRISM® 7000 Sequence Detection System) to evaluate GAPDH expression using SYBR® Green and GAPDH-specific primers .
- Begin by preparing RNA from tissue according to Ambion's ToTALLY RNA™ protocol
- Save the organic phases, including the interfaces, from the Solution 1 and Solution 2 extractions.
- Combine the reserved organic phases in a vessel large enough that an equal volume of extraction buffer can be added with some room left over.
- Prepare an equal volume of extraction buffer:
|10 mM||Tris-HCl pH 8.0|
- Adjust to pH 12.0 with 5 N NaOH immediately prior to use with pH paper or a pH meter.
- Add to the combined organic phases. Shake well for 1 minute. Place on ice for 10 minutes.
- Centrifuge for 20 minutes at 10K, 4°C
- Recover the aqueous phase, leaving behind any interface. Add 1/15 volume 7.5 M NH4OAc followed by 2 volumes ice-cold 100% EtOH. Incubate at -20°C for at least 1 hour.
- Centrifuge for 20 minutes at 10K, 4°C
- Carefully decant the supernatant. The pellet may not be visible at this point.
- Gently add 1 ml of 70% EtOH to rinse the pellet. Swirl the vessel gently. Centrifuge briefly to ensure that the pellet remains attached. Carefully pour off the supernatant. Use caution as the pellet may not be visible at this time.
- Allow pellet to dry at room temperature until no liquid is visible in the vessel.
- Suspend the DNA pellet using TE (10 mM Tris-HCl, pH 8.0 / 0.1 mM EDTA, pH 8.0) in approximately 1/2 the volume required for the RNA recovered - for a first approximation use 25 to 50 µL TE per 100 mg of starting material. Heat 5 minutes at 55°C then vortex thoroughly to dissolve the DNA.