The following information should act as a guide for important steps in performing a quantitative experiment. It is not intended to cover every aspect of an experiment but rather to give suggestions for the major steps involved.
Consultation and careful planning in the beginning will prevent ruining your samples for the whole experiment. Important aspects to monitor so this doesn’t happen are the collection, handling and storage of the samples. This is particularly true in studies involving human patients or animal studies where samples are collected in a clinic or operating room.
Nucleic Acids: it is critical that tissue samples be stabilized in terms of RNase activity immediately following their isolation from the host. RNase is a small, very active enzyme that cannot be inactivated even by autoclaving. All methods of RNase stabilization inactivate enzymatic activity but the enzyme will come back to life (refold into an active state) easily when conditions become favorable again. RNase will continue to work, albeit slowly, at -80(degree symbol here)C. Although DNA degradation can be a problem as well, DNase requires some Ca+2 as a co-factor for activity and is not nearly as robust as RNase. The most common mammalian RNases do not require a co-factor for enzymatic activity. DNA hydrolysis can be more easily stopped by including EDTA in the lysis buffer.
Most importantly, do NOT let the tissue sample linger at room temperature following isolation from the organism of origin. The nucleic acids will begin to breakdown immediately following removal from the organism, especially the RNA. The breakdown process is rapid and thorough. A good way to avoid this, when it is not possible to process samples immediately upon collection is to use a product called RNAlater (Qiagen or Ambion). This solution will stabilize the RNA within the tissue at room temperature for a few hours or at 40°C or -20°C for longer periods of time. Large tissue samples need to be cut into thin slices, consult the manual for directions on proper handling.
Proteins: the storage of tissue for protein isolation will be dependent on the down stream experimental design. However, as with nucleic acids, it is critical that tissue samples be handled such that the proteins of interested will be stabilized as soon as possible following sample collection from the source. This may involve the addition of protease inhibitors, reducing agent etc, and rapid storage at -80°C.
Nucleic Acids: There are 3 choices for inactivating RNase during RNA isolation: 1- if your tissue is small or can be cut into thin slices, RNAlater is a good choice for stabilizing the RNA in the tissue as mentioned above; 2- tissue can be added to a guanidinium saturated phenol solution such as Trisol, TriReagent, RNAsol, etc. and homogenized immediately following extraction from the subject using either a Polytron, mini-homogenizer or bead beater. The resulting homogenate can be stored at -80°C for long periods of time prior to the isolation of RNA or DNA and 3- the tissue can be flash frozen in liquid nitrogen and stored at -80°C. For some tissues, where a LN2 frozen powder is required prior to nucleic acid isolation, this may be preferable. A phenol-based reagent is recommended for the final nucleic acid isolation here as well.
Regardless of which of the above three methods are employed, it is important to couple the homogenization process to a final column-based RNA isolation step. Using the homogenization reagent alone will carry over a lot of DNA and may not remove all the proteins, RNA or DNA binding proteins in particular, on the first pass. Using a column will get rid of these carryover contaminants.
For tissue culture cells, RNAlater can be used if there needs to be a delay in nucleic acid isolation or for immediate isolation a guanidinium lysis buffer without phenol can be used if desired but again it should be followed by a column-based clean up.
For RT-PCR or PCR, another approach is to not isolate the nucleic acids at all but rather to make a cell or tissue lysate that you can use directly in an RT reaction or in a PCR. Most tissue culture cell lines can be lysed directly in Cells to Ct reagent from Life Technologies, or the RTR Cell Lysis buffer from Roche. Although a new application of the Cells-to-Ct product, you can try homogenizing small tissue samples in this reagent with a homogenizer or bead beater. A third a novel product is RNA GEM from ZyGEM which digests all the proteins in the lysate rapidly leaving behind clean RNA and DNA. These methods work well and saves the cost of isolating RNA. Further, there is no loss of any of the contents of the tissue or cells during macromolecule isolation.
For isolation of microbial or viral nucleic acids from mammalian sources, it is best to try to remove the abundant mammalian contamination from the usually minimal microbial target without lysing the microbe. This can be accomplished by differential lysis of the mammalian cells while leaving the microbial cells intact. Isolation of total nucleic acids from all sources within the sample will result in a needle in a haystack problem for the detection of the microbial target. The isolation of viral DNA or RNA away from the host can be more problematic unless the majority of the viral particles are extra-cellular.
Proteins: There are a large number of buffers in the literature for the isolation of different proteins from multiple sources, too many possible combinations to cover in this space. Purify your protein as per your standard protocol or one from the literature.
Handling and Storage
Nucleic Acids: Although not as sensitive to freeze/thaw cycles as proteins, it is not a good idea to freeze and thaw nucleic acid samples too many times. If possible, aliquot them early on and at a concentration that is high enough to perform any assay requiring high concentrations. Further, nucleic acids store better at higher concentrations (e.g., 0.25 – 1.0 µg/µl). It’s a lot easier to dilute samples for one assay than to concentrate them later. Perform all the QC analyses required on the initial sample such as an estimate of concentration and cleanliness by absorbance: 1 OD = 40 µg/ml for RNA; 50 µg/ml for DNA; A260/A280 1.8 (max is 2.0) or better for DNA; 1.9 (max is 2.2) or better for RNA) and A260/A230 (1.8, preferably 2.0 for both) and Bioanalyzer (Agilent 2100) or Experion (Bio-Rad) chip analysis, if necessary, to determine structural integrity of RNA or small DNAs. Based on the concentration determined from absorbance, dilute each sample to a common final concentration (TE for DNA, nuclease-free H2O for RNA) and make aliquots of each sample and store in the freezer. For submitting samples to the QGCL for processing, samples should be placed into 1.5 ml screw cap tubes (ISC Bioexpress cat# C-3295-2). The tube geometry is critical for correct liquid handling by the robot, no substitutions can be made. RNA samples should be DNase I treated prior to submission to ensure that the DNA background is minimal at best. An easy protocol is available for download here.
Proteins: The most important thing to consider once you have freshly isolated protein is how many aliquots do I need to make, how much volume per aliquot and what vessel to use for the aliquots? Some consideration must be made prior to protein isolation on how and where they will be stored prior to use. For ELISAs, more than 2 freeze thaw cycles is not recommended (chemokines and cytokines, in particular) and this is the case for many other classes of proteins as well. Samples should be centrifuged to remove any particulates. If your target protein(s) is in low abundance, you might consider using siliconized storage tubes to prevent loss on the tube walls. A carrier such as 1% final BSA has been recommended for some sample types (e.g., saliva, bronchoalveolar lavage fluid and urine-plus buffer) and applications (ELISAs). However, you would not want to add BSA if you planned to use the samples for Mass Spectrometry analysis, for example. Determine the concentration of your proteins, dilute to a common concentration in the appropriate buffer for downstream analyses and aliquot into the chosen vessels. When preparing tissue or tissue culture cell material, keep the SDS and reducing agent levels low as they can affect the ability of Abs to work properly. Store aliquots of your protein samples at -80°C until used. If proteins are to be used for Meso Scale quantitative ELISAs and processed within the QGCL, sample aliquots should be stored in the appropriate 96-well plate for automated processing. Check with the core lab director for more information.