Female wild-type C57BL/6J mice (9–10 weeks old, 18–22 g) and BALB/c nude mice (7–8 weeks old, 18–20 g) were obtained from the Animal Center of Peking University Health Science Center. All procedures were approved by the Institutional Animal Care and Use Committee of Peking University (PUIRB-LA2023330). All the methods and experimentation were performed according to the relevant regulations and guidelines, including the ARRIVE guidelines. Mice were housed under standard conditions with ad libitum access to food and water. After a two-day acclimatization period, mice were euthanized through cervical dislocation, and the following tissues were collected: bone (from the tibia and femur excluding bone marrow), colorectum, heart, kidney, liver, lung, lymph node (from the inguinal and axillary regions), muscle (from the gastrocnemius and tibialis anterior muscles of the leg), ovary, skin, spleen, stomach, thymus, and uterus from C57BL/6J mice; since the thymus and lymph nodes are absent or minimally developed in BALB/c nude mice, only the other twelve types of tissue were collected. Cervical dislocation was performed without prior anesthesia, as the use of anesthetics may interfere with downstream molecular analyses; this method is accepted for rapid euthanasia by trained personnel (AVMA Guidelines for the Euthanasia of Animals: 2020 Edition). Tissue weights ranged from 6 to 45 mg. Larger tissues were divided into small pieces and homogenized using a Mixer Mill (Retsch, 30 Hz, 3 min) in appropriate lysis buffer until no visible lumps remained.

DNA and RNA were extracted from separate tissue aliquots using the QIAamp DNA Mini Kit and RNeasy Plus Universal Mini Kit (Qiagen), respectively. All procedures were performed strictly in accordance with the manufacturer’s instructions using manual procedures performed individually per analyte. Elution volumes were 50 μL for DNA and 40 μL for RNA. Concentration and purity (260/280 nm ratio) were determined using a NanoDrop™ One spectrophotometer. DNA integrity was evaluated by agarose gel electrophoresis; approximately 1 μg of genomic DNA was mixed with 2 μL of 0.25% bromophenol blue solution and separated at 16 V/cm for 30 min on a 1% agarose gel in Tris-borate-EDTA (TBE) buffer. DNA bands were visualized under ultraviolet (UV) light and photographed using the Tanon System. RNA quality was assessed using capillary electrophoresis (Fragment Analyzer 5200).

For protein extraction, tissues (except bone) were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) supplemented with Protease Inhibitor Cocktail (AMRESCO), incubated on ice for 30 min, and centrifuged at 13,600 g for 20 min at 4 °C. The supernatant was collected as total protein, as previously reported (Chen et al., 2025). Bone tissue was processed using a Bone Tissue Protein Extraction Kit (Aidisheng): Fresh bone tissue samples were soaked in phosphate-buffered saline (PBS) buffer at 4 °C twice. The bones were then washed with distilled water to remove any residual red blood cells. The bone tissue was cut into small pieces, weighed, and placed into a mortar filled with liquid nitrogen, where it was ground into a fine powder. 500 μL of Protein Extract A, along with 2 μL of protease inhibitor and 2 μL of protein stabilizer, was added to 200 mg of bone tissue. The mixture was shaken at 4 °C for 30 min and subjected to ultrasound at 80–100 W for 20 cycles in an ice bath (5s ultrasound/5s interval). Subsequently, the mixture was centrifuged at 4 °C at 12,000 g for 10 min. The supernatant was collected as the total protein extract from the bone tissue. Protein concentrations were measured using the bicinchoninic acid (BCA) Protein Assay kit (Thermo Scientific). A total of 25 μL of protein sample and 200 μL of BCA working solution were added to a 96-well plate, followed by incubation at 37 °C for 30 min. The absorbance was then measured at 570 nm using a microplate reader (Tecan). A standard curve (y = 1.2416x - 0.1685, R² = 0.996) was generated based on the absorbance values of various concentrations of bovine serum protein standards (0 μg/mL, 25 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, 750 μg/mL, 1,000 μg/mL, 1,500 μg/mL, and 2000 μg/mL).

For each tissue type, six biological replicates (n = 6 per strain) were used. Data are now presented as mean ± standard deviation. Data were analyzed using GraphPad Prism 9.3.0. One-way ANOVA with post hoc tests (Newman-Keuls, Tukey, or Kruskal-Wallis) was used for multiple comparisons. A p-value <0.05 was considered significant.

DNA was extracted from mouse tissues of various organs, including bone, colorectum, heart, kidney, liver, lung, lymph node, muscle, ovary, skin, spleen, stomach, thymus, and uterus. The optical density (OD) ratios at 260/280 nm of DNA from each group were measured using a NanoDrop™ One microvolume spectrophotometer (Figure 1A). In C57BL/6J mice, the average 260/280 ratios across the fourteen tissue groups ranged from 1.91 ± 0.03 to 2.02 ± 0.04, with no statistically significant differences among groups (P > 0.05). Similarly, in BALB/c nude mice, the average ratios across twelve groups ranged from 2.00 ± 0.06 to 2.13 ± 0.02, also with no significant intergroup differences (P > 0.05). Nearly all DNA samples exhibited purity within the accepted range of 1.8–2.1 (Ahlberg et al., 2021; Qian et al., 2017), indicating that high-purity DNA can be consistently extracted from different organ types using the same method.

DNA yields were compared across organ types. Concentrations were measured using the NanoDrop™ One, and yields were calculated based on the tissue weight used for extraction as previously studies did by normalizing yields to tissue weight (Hofstetter et al., 1997; Pearce et al., 2024; Austin et al., 2016; Stroh et al., 2021; Zeng et al., 2024; Siuta et al., 2023) (Figure 1B; Table 1). Yields were normalized to tissue weight by dividing the total amount of DNA (µg) by the tissue mass (mg) used for extraction. In C57BL/6J mice, the highest DNA yields were obtained from kidney (1.98 ± 0.33 μg/mg), spleen (2.03 ± 0.26 μg/mg), and colorectum (2.45 ± 0.61 μg/mg), while the lowest yields were from bone (0.22 ± 0.07 μg/mg) and muscle (0.38 ± 0.12 μg/mg). In BALB/c nude mice, the highest yields came from colorectum (4.50 ± 1.19 μg/mg), stomach (4.60 ± 0.69 μg/mg), and uterus (3.82 ± 0.71 μg/mg), and the lowest from bone (0.51 ± 0.13 μg/mg), heart (0.76 ± 0.14 μg/mg), and muscle (0.59 ± 0.05 μg/mg). Coefficients of variation (CV%) for DNA yields per organ type are presented in Table 4.

DNA quality was evaluated by gel electrophoresis (Figures 1C,D). Most extracts showed intact bands around 20 kb, though mild degradation was observed in DNA from colorectum, spleen, stomach, and thymus. These findings demonstrate that DNA yield varies by tissue type when using a standardized extraction protocol.

To facilitate biobank sample handling, we determined the amount of tissue needed to obtain 10 μg of DNA, a typical requirement for applications such as PCR (While a single qPCR reaction requires only 10–100 ng of DNA, the total amount needed for a comprehensive experiment (including multiple targets, replicates, and standard curves) can reach 10–100 µg) and whole-genome sequencing (WGS). Correlation analysis confirmed a positive relationship between starting tissue weight and DNA yield within the same tissue type using the same extraction method (Supplementary Figure S1A). The tissue masses required to extract 10 μg DNA are then calculated and summarized in Table 5. Since weighing tissue manually is often impractical, we also provided visual references comparing tissue sizes to common seeds (e.g., sesame, rice, mung bean; Figure 2; Supplementary Figures S1B–M). For example, in C57BL/6J mice, 5.71 mg of thymus is comparable to a sesame seed, and 9.08 mg of lymph node tissue approximates three lymph nodes, each smaller than a sesame seed.

RNA was extracted from the same set of organ types. The 260/280 nm ratios were measured using a NanoDrop™ One (Figure 3A). In C57BL/6J mice, average ratios per group ranged from 1.95 ± 0.14 to 2.08 ± 0.03; in BALB/c nude mice, they ranged from 1.88 ± 0.09 to 2.10 ± 0.03. No significant differences were found among groups (P > 0.05). Nearly all values fell within the accepted purity range of 1.8–2.1 (Ahlberg et al., 2021; Qian et al., 2017), confirming that the extraction method consistently yields high-purity RNA across organ types.

RNA concentrations were measured, and yields were normalized to tissue weight by dividing the total amount of RNA (µg) by the tissue mass (mg) used for extraction (Figure 3B; Table 2). In C57BL/6J mice, the highest RNA yields were from colorectum (3.46 ± 0.28 μg/mg), kidney (2.96 ± 0.39 μg/mg), and liver (2.80 ± 0.50 μg/mg), while the lowest were from bone (0.16 ± 0.05 μg/mg), muscle (0.34 ± 0.10 μg/mg), and skin (0.25 ± 0.07 μg/mg). In BALB/c nude mice, the highest yields were from spleen (3.79 ± 0.73 μg/mg), liver (2.63 ± 0.71 μg/mg), and colorectum (2.24 ± 0.65 μg/mg), and the lowest from bone (0.13 ± 0.06 μg/mg), muscle (0.37 ± 0.07 μg/mg), and skin (0.53 ± 0.18 μg/mg). CV values are provided in Table 4.

RNA quality was assessed by capillary electrophoresis (Figure 3C; Supplementary Figures S2, S3). The RNA quality number (RNA quality number (RQN)) ranged from 6 to 10 across all organ types except stomach, indicating good integrity. Thus, RNA yield also varies substantially across tissues under standardized extraction conditions.

We next determined the tissue mass required to obtain 10 μg of RNA, a common amount needed for real-time PCR or RNA-seq. A positive correlation was observed between tissue input and RNA yield within the same tissue group using the same extraction method (Supplementary Figure S4A). The tissue masses required to extract 10 μg RNA are then calculated and summarized in Table 5. Visual size comparisons using common seeds are provided in Figure 4 and Supplementary Figures S4B–M. For instance, in C57BL/6J mice, 6.73 mg of lung tissue is roughly equivalent to a sesame seed, and 14.17 mg of lymph node tissue corresponds to four lymph nodes, each smaller than a sesame seed.

Total protein was extracted from all organ types using RIPA buffer (or a specialized kit for bone). Protein concentration was determined via BCA assay, and yields were normalized to tissue weight by dividing the total amount of protein (µg) by the tissue mass (mg) used for extraction. (Figure 5A; Table 3). In C57BL/6J mice, the highest protein yields were from kidney (124.39 ± 12.06 μg/mg) and liver (120.18 ± 8.44 μg/mg), and the lowest from bone (18.69 ± 5.91 μg/mg). In BALB/c nude mice, the highest yields were from liver (192.31 ± 4.90 μg/mg), lung (169.50 ± 8.83 μg/mg), and heart (168.10 ± 15.89 μg/mg), and the lowest from bone (43.34 ± 17.22 μg/mg). Coefficients of variation (CV%) for protein yields per organ type are presented in Table 4. These results indicate that protein yield is also highly tissue-dependent when using the same extraction method.

We calculated the amounts of tissue from various organ types required to extract a specific quantity of protein. We first conducted the correlation analysis using data from the same tissue group and the results indicated that the yields of protein exhibited a positive correlation with the weight of the starting material (Supplementary Figure S5A). Based on the results of correlation analysis, we calculated the amount of tissue needed to obtain 1 mg of protein (Table 5). Size comparisons are illustrated in Figures 5B–O and Supplementary Figures 5B–M. For example, in C57BL/6J mice, 14.29 mg of thymus is similar in size to a grain of rice, and 23.07 mg of lymph node tissue corresponds to seven lymph nodes, each smaller than a sesame seed.

Finally, we analyzed the correlation between DNA, RNA, and protein yields of C57BL/6J mice and BALB/c nude mice based on the weight-normalized median data points per organ (Figures 6A–C). The results showed a weak (DNA) or strong (RNA and protein) positive correlation in the yields between the two strains, suggesting similarity at least in the RNA and protein yields of the two strains.