What types of samples can be analyzed?
Most types of biological specimens can be analyzed. However, there are some samples that cannot be analyzed depending on the condition of the sample or the sample preprocessing method. Please refer to the Q&As below.
Are there any samples that cannot be analyzed?
Samples that are difficult to analyze include those that render poor extraction of metabolites and those with large bias in the concentration of the extracted metabolites.
The former refers to samples with very high viscosity or hard samples such as seeds and bones. However, if you pulverize and extract the metabolites on your own, we might be able to measure such samples.
The latter refers to samples with very high salt concentrations, such as seawater and beverages that contain very high concentrations of certain metabolites. In addition, it may be difficult to analyze samples with significantly different pH levels in the same test. Since these samples are diluted during measurement, it will be hard to detect other metabolites with relatively low concentrations and the number of detected metabolites may be significantly reduced.
Are there any sample types that require special attention?
The main points to note are listed for each type of sample; please check the relevant sample type below.
Samples of human origin
For human samples, please confirm that there are no viruses or microorganisms present in the samples before sending them to us.
If you wish to analyze samples that are suspected of being infected, or confirmed to be infected (i.e., infectious disease samples), you will need to perform deproteinization and/or other relevant treatments for these samples.
Please note that you will need to provide information about the disease and anonymity of the specimen.
Although we have experience in analyzing whole blood, plasma, and serum samples at HMT, we recommend plasma samples for metabolome analysis.
This is because for whole blood samples, the effect of metabolites derived from blood cell components must be taken into consideration, whereas for serum samples, metabolite levels may fluctuate depending on the incubation time and environment.
However, if you have used whole blood or serum in a previous experiment, we recommend using the same conditions for the current study.
Additionally, EDTA is recommended as an anti-coagulant for the preparation of plasma samples.
Basic measurements can be performed without any issues, however, in such samples that do not contain cells, metabolites that are considered to be present in small amounts outside of cells, such as glucose-6-phosphate, tend to be difficult to detect.
For this reason, the number of detected metabolites is expected to be lower in the analysis of energy metabolism pathways i.e., C-SCOPE. Therefore, please consider carefully whether the analysis plan is in line with your objectives.
In principle, plant samples, including vegetables and fruits, can be analyzed. However, plant samples are often subject to Japan’s customs restrictions, so please contact us if you would like to analyze any plant sample and kindly provide the relevant information (e.g., species, genus, type (leaf, stem, fruit, etc.) in your inquiry.
Since the amount of metabolites significantly differs depending on the parts of a plant, it is important to consider which part of the plant is used for the test and how to handle the corrected weight, including the water content.
For samples that are difficult to homogenize at HMT e.g., roots and seeds, you may be asked to perform the metabolite extraction.
Fermented seasonings, extracts, etc.
It is generally possible to analyze without any issues, however, for samples with high salt concentration, high viscosity or high levels of specific metabolites, the number of detected metabolites may decrease due to sample dilution (prior to analysis).
What is the required sample volume/mass?
The amount of sample required depends on the analysis platforms (CE-MS, LC-MS).
As an example, the required sample volume/mass for analysis using CE-MS (Basic Scan, C-SCOPE, etc.) are listed below. (It is also possible to analyze samples with an amount smaller than indicated in the table, so please consult with us.)
|Required sample volume/mass
(For CE-MS analysis)
|20 - 40 mg
|1.0 - 5.0×106 cells
(cells with small diameter)
|1.0 - 5.0×107 cells
(cells with large diameter)
|20 - 50 mg
|1,000 μL (1 mL)
Please note that the sample volume is important for normalization so be sure to weigh/measure accurately.
What does “/OD600” mean?
“/OD600”means (OD600 value) x mL. For example, “20/OD600” means that if the OD600 value of your sample is 0.5, 40mL of bacterial culture is required. If the OD600 value is 2.0, 10mL is required.
Difficulty in collecting the required amount of sample
In some cases, it may be possible to analyze samples with the amount that you are able to prepare so please feel free to consult us.
For mouse tissues, it may be useful to collect samples from multiple subjects and consider them as one sample.
What does “normalization value” mean?
At HMT, metabolites are extracted and measured in a fixed volume of solution regardless of the original sample volume. The normalization value is calculated using the original sample volume to calculate the amount of metabolite per unit volume.
Hence, if the normalization is inaccurate, it will be difficult to discern whether the apparent difference in the amount of metabolites is due to the variations between samples, or due to the sample amount.
For animal and plant tissues and cultured cell samples in particular, accurate measurements of the weight, cell count, etc. are required for normalization. Additionally, we recommend freeze-drying in advance if the water content in the sample is expected to vary significantly, since variations in water content may affect the weight.
Moreover, for cultured cells, if the cell size differs depending on the cell type or due to drug treatment, we recommend using protein content or other normalization factors instead of cell number.
What should I do if the weight of my sample cannot be determined?
Weighing of samples at HMT can be provided for a fee.
For frozen samples, please weigh them before sending them to us in order to avoid freeze-thaw cycles.
Excision and dispensing of sample can also be provided for a fee (only for non-human derived samples); however, specifying the part of the sample is not possible.
What is the required sample size (n number)?
At HMT, the recommended number of specimens per group is 1-3 for cultured cells and microorganisms, 3-10 for experimental animals and cultivated plants, and >20 for clinical specimens, and wild plants and animals.
For cultured cells and microorganisms, n=1 may be sufficient if the heterogeneity of the cell population can be eliminated but n=3 may be useful when verifying drug response in cultured animals or plants. However, for simple comparison analysis, n=5 to 10 is recommended.
For clinical specimens, wild plants and animals, n=20 is ideal to evaluate individual differences. If the symptoms are distinguishable, n=20 is ideal, but if the heterogeneity within a group (e.g., symptoms differ among individuals with mental disorders) is considered, n=50 would be ideal. In any case, randomization should be performed to eliminate as many confounding factors as possible, such as age, gender, and BMI.
For any sample type, the reviewers might address the sample size in your study design during manuscript submission. Therefore, we recommend that you refer to the sample sizes used in previous studies.
If you have any questions regarding study design, please contact us.
Is it possible to perform multiple analyses?
Metabolite amounts are reported as “relative area values” for most analysis plans. Since relative area values do not have units, it is not possible to make a comparison if these values are derived from different studies.
HMT’s analysis plans that report quantitative values, such as C-SCOPE, and Q-OPTION 110, measures a certain concentration of the analyte prior to measurement and creates a calibration curve based on these values to calculate the concentration. This allows data integration and comparison between multiple analyses.
However, please be advised that there are some factors that may make it difficult to integrate the data from multiple analyses, such as inconsistencies or measurement errors during sample preparation, and degradation of metabolites during storage.
How should I store and ship the samples?
Samples that can be stored at room temperature or under refrigeration (e.g., food) should be stored under those conditions, while other samples should be stored at –80℃.
Shipping of samples to HMT will be handled by our authorized distributors and the relevant instructions for sample preparation and shipping will be provided upon order confirmation. If your distributor is not listed here, please contact HMT.