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A successful experiment starts with a good experiment design. Based on our extensive experience in analysis of nucleic acids, we recommend to take the following considerations into account in the design of your experiment:
Eliminate variances in materials, procedures, equipment, and personnel. Examples include using a single lot of reagents and materials, standardization of all labware, and ensuring batch, operator or time point are characterized and variability minimized. In addition, samples should be collected under similar conditions and where possible at the same time, relative to e.g. treatment or food intake. If elimination of these sources of variation is not feasible, ensure these “noise” factors are built into your experimental design. At Biogazelle, a project manager will assist you in identifying possible sources of variation and can support you in optimizing your experiment design to cope with the inherent noise factors.
The number of samples required depends on various factors, such as the research question, the data analysis strategy, the magnitude of the measurement difference, and the measurement variability within each experimental group. The latter is inherent to the underlying biology, collection, isolation, and analysis. A pilot study may be required to quantify this variability and provide an educated estimate of the number of samples required.
We recommend an absolute minimum of three biological replicates per group for hypothesis testing: for cultured cells, at least 3 independent replicates should be included; for inbred organisms (e.g. lab mouse) at least 6 animals per group are advised; for human individuals at least 12 individuals per group are recommended. In general, to generate robust results, the rule “the more samples per group, the better” applies. As such, for a biomarker study, a typical starting number when working with human individuals, is 30 samples per group.
Conduct a study on representative samples to ensure your sampling method maintains the representation, quality, and yield of the RNA or DNA required to draw valid conclusions. This often requires a careful pilot study prior to the main study. Remember, garbage in equals garbage out!
If a customer provides this information, it will be reported back along with your experimental results so you may track isolation method performance against experimental results. It can also assist the Biogazelle project manager in troubleshooting anomalous results if they occur.
At Biogazelle, we have accumulated considerable experience processing liquid biopsies from different sources. Below, we provide recommendations for the most common body fluids. For specific instructions on less common liquid biopsies, take a look at the general recommendations at the top of the list, or contact one of our experts.
At Biogazelle, we use validated, ISO17025- and GCLP-compliant procedures for nucleic acid extraction from liquid biopsies. These procedures have been carefully selected to suit the downstream applications offered by us.
Should you prefer to perform nucleic acid extraction at your site, we recommend to take following considerations into account:
Several commercial kits for nucleic acid extraction from body fluids are available. However, we recommend to use following extraction methods as they have been extensively tested and used in Biogazelle’s downstream applications: miRNeasy serum/plasma kit from Qiagen for RNA extraction and the QiaAmp circulating nucleic acid kit from Qiagen for DNA extraction.
The volume of liquid biopsy used for nucleic acid extraction will directly impact the yield. Therefore, increasing the liquid biopsy input volume is advised if large amounts of nucleic acid are required. The workflows at Biogazelle are optimized to reach maximum sensitivity with low RNA or DNA input amounts. Therefore, we recommend to extract RNA from 0.2 ml and DNA from 2 mL of serum or plasma. For specific instructions on other body fluids, ask one of our experts.
Nucleic acid extraction from liquid biopsies typically results in low amounts of fragmented RNA or DNA. As such RNA/DNA concentration measurements are generally not reliable and we standardize our workflows based on volume input. Furthermore, we have optimized our workflows to achieve maximum sensitivity on minute amounts of fragmented nucleic acids. As a result, the concentration and integrity of RNA or DNA extracted from liquid biopsies should not be assessed prior to analysis at Biogazelle.
Cell-free RNA may contain traces of genomic DNA that could interfere with some downstream analyses. Therefore, we recommend to perform a DNase treatment on purified RNA, if required by the downstream application. Reach out to our the experts to learn which applications require removal of gDNA.
Upon careful sample collection according to standard operating procedures and following a suitable experiment design, your samples are ready for shipment to Biogazelle. To preserve the quality of your samples, we recommend to take following considerations into account when preparing your shipment:
If shipping more than 48 RNA or DNA samples: