Rectal swab DNA collection protocol for PCR genotyping in rats¹

Abstract

DNA collection is essential for genotyping laboratory animals. Common collection methods require tissue amputation, causing discomfort and injury. Rectal swabbing has been proposed as an effective, minimally invasive alternative, but an evidence-backed protocol for the technique remains unavailable. This report evaluates the effects of collection parameters on the quality of PCR results and presents a protocol for genotyping a litter of rats within 3–5 h. Samples with 2–8 scrapes produced enough DNA to amplify targets up to ∼1800 bp long using PCR. Rectal swabbing produced PCR results with similar utility as ear clip samples, and results were unaffected by residual fecal matter or cell debris. The protocol enables fast, minimally invasive and repeatable genotyping using commercial PCR reagents.

Plain language summary

DNA collection for genotyping laboratory rodents commonly requires tissue amputation, leading to injury and discomfort. Rectal swabbing can be an effective, minimally invasive alternative, but there is a lack of information on how to apply the technique and the necessary parameters involved. This report assesses collection and processing parameters while combining the rectal swab collection method with commercial PCR reagents to allow for fast genotyping with minimally processed DNA samples. Findings were used to design a protocol for minimally invasive DNA collection and genotyping.

Tweetable abstract

Rectal swabbing is an effective yet minimally invasive technique for collecting DNA from laboratory rats. This new protocol consistently produces PCR-quality DNA while remaining repeatable and cost effective. #Genotyping #PCR #Transgenic

Method summary

Various parameters of the rectal swabbing technique for DNA collection were evaluated. These parameters included the number of rectal scrapes, number of samples, and contamination with fecal matter or cell debris. Samples with 2–8 scrapes yielded sufficient DNA for PCR amplification of genomic sequences up to ∼1800 bp. Contamination with fecal matter or cell debris did not affect PCR outcome quality. Rectal swabbing produced similar results across rats of both sexes and different ages.

Executive summary

• Common DNA collection techniques for genotyping laboratory rodents require tissue amputation, causing injury and discomfort.

• Rectal swabbing can be an effective, minimally invasive alternative but is not commonly used.

• There is no existing protocol for performing the procedure.

Experimental

• Rectal swab collection was conducted with genetically modified male and female rats of various ages.

• The number of samples, number of rectal scrapes per sample and presence of fecal matter or cell debris were experimentally varied to determine their effect on PCR amplification.

Results & discussion

• A total of 2–8 scrapes of the rectal epithelium can yield sufficient DNA for the PCR amplification of genomic targets up to ∼1800 bp.

• Fecal matter and cell debris do not affect PCR outcome quality.

• A detailed protocol for this procedure is provided.

Conclusion

• Rectal swabbing is an effective and minimally invasive alternative for DNA collection in rats.

Keywords: :

• DNA collection
• DNA collection protocol
• genotyping
• minimally invasive
• PCR analysis
• polymerase chain reactions
• rectal swab
• rectum swab
• sampling
• transgenic rats

Collecting DNA is essential for genotyping transgenic animal models. Many collection techniques provide DNA samples that can be amplified and analyzed with PCR. These techniques vary in invasiveness, repeatability and ease of processing. The most commonly used techniques include ear, tail and distal phalanx clipping [1], which yield high quantities of DNA but are invasive. Clipping involves the removal or partial amputation of body appendages, causing injury and often requiring a recovery period and local or general anesthetics to reduce pain [^2-4]. Tail and distal phalanx clipping both have age-related limitations as well, relying on small time windows for sample collection and genotyping [^4-7]. Because of their invasiveness and risk for injury, repeated collection with these techniques is either discouraged or not permitted [2,5,8,9]. Alternatively, noninvasive and minimally invasive techniques such as fecal pellet collection, oral swabbing and hair follicle collection [5] can be effective and noninjurious. While they avoid surgical incisions, they too pose unique challenges. Fecal pellet collection can be more time-consuming than other minimally invasive methods [5,10,11], oral swabbing has a lower expected DNA yield [5] and hair follicle samples can cling to equipment, increasing the risk of cross-contamination [12].

Rectal swabbing [13] is a promising alternative, minimally invasive collection technique. This method was reported to be quick, easily repeatable and effective for genotyping both weanlings and adult mice [13]. However, details of the technique could not be located in the original report or in the few subsequent published works [5,12]. Thus, there is a lack of information on how to apply the rectal swab technique for DNA collection and understanding of the critical parameters that determine its success. In this report, the authors evaluated how collection parameters, such as scrape number, sample number and fecal matter or cell debris contamination, affected the genotyping outcome for two genomic targets of different lengths: Pvalb^Cre (1803 bp) [14] and Sox21 (237 bp). The findings informed the creation of a new rectal swab genotyping protocol that repurposes a commercially available PCR kit for quick collection, DNA extraction and PCR. With this protocol, sample collection and PCR genotyping for an average litter of rats can be completed within 3–5 h. Rectal swabbing is an effective, minimally invasive and repeatable DNA collection technique for PCR genotyping.

Materials & methods

Experimental rats & materials

The rectal swab genotyping technique was evaluated using 21 heterozygous transgenic Pvalb^Cre rats (12 males and 9 females) [14] of different ages (6, 15, 17, 27, 28, and 42 weeks). Each animal was used for multiple collections. DNA extraction and PCRs were performed using the Thermo Scientific™ Phire Tissue Direct PCR Master Mix (Thermo Fisher Scientific #F-170S, MA, USA). The developed protocol for rectal swabcollection and genotyping is available as Supplemental data.

Rectal swabbing

Rats were gently restrained and a sterile 1-μl inoculation loop (Thermo Fisher Scientific 22-363-601) was inserted 2 mm into the rat's rectum. The loop was used to carefully scrape the rectal lining and collect epithelial cells (Figure 1). The researcher waited for any droppings to exit the rectum before inserting the inoculation loop and repeated collection if there was visible fecal matter on the loop. The tip of the loop was then briefly swirled and clipped off into a 0.2-ml tube pre-filled with 20 μl of dilution buffer from the Phire Tissue Direct PCR kit.

Following the manufacturer's instructions [15], 0.5 μl of DNA Release Additive included in the kit was added to each tube before briefly vortexing and centrifuging. The samples were incubated at room temperature (∼20°C) for 2–5 min, heated to 98°C for 2 min and then centrifuged for ∼15 s to separate cellular debris from supernatant. A total of 13 μl of supernatant was withdrawn and stored (-–20°C), 1 μl of which was used for PCR.

Experimental variations

The proposed parameters of scrape number, fecal matter and cell debris were varied to test how they each affect the outcome of the genotyping procedure. To test the effect of scrape number, samples were collected with various scrape numbers (2, 4, 8, 12, 16 and 20) from 12 rats (7 male, 5 female). Samples were collected across 8 days while taking a maximum of 20 scrapes from each rat in a given day. This spacing was done to reduce the potential adverse effects of excessive rectal scraping beyond realistic collection demands. A total of 16 scrapes per sample were used for subsequent experiments to maintain consistency. To determine the optimal number of samples per rat, three additional samples from each rat were collected and analyzed. To test the effect of fecal matter, one additional sample with visible fecal matter on the inoculation loop was collected from 6 rats (4 male and 2 female). To test the effect of cellular debris, another sample from was collected from these 6 rats but was not centrifuged to remove debris from the supernatant during DNA extraction.

Ear clipping

A piece of tissue was clipped from 6 experimental rats' ears (3 males, 3 females) using a 0.5-mm puncher. DNA was extracted using the same procedure and reagents as for the rectal swab samples.

Oral swabbing

Oral swabbing was conducted on 9 experimental rats (5 males, 4 females) using a 1-μl inoculation loop. The rats were restrained with one hand and the inoculation loop inserted into its mouth with the other hand, gently scraping the buccal cavity 4–5 times. DNA was extracted for oral swab samples using the same procedure and reagents as described for rectal swab samples.

PCR

The Pvalb^Cre product (1803 bp) was amplified using forward primer sequence: 5′-GTCATGAACTATATCCGTAACCTGG-3′ and reverse primer sequence: 5′- AGTGGTGCACACCCTGATAC-3′ with final concentrations of 0.5 μM. These were designed for the current study. The Sox21 product (237 bp) was amplified using the forward and reverse primers: 5′-AGCCCTTGGGGASTTGAATTGCTG-3′ and 5′- GCACTCCAGAGGACAGCRGTGTCAATA-3′, respectively (manufacture supplied), at a final concentration of 0.125 μM. The following cycling protocol was used on a Bio-Rad T100 Thermal Cycler (California, USA): 98°C for 5 min, followed by 35 cycles of 98°C for 5 s, 64°C for 5 s and 72°C for 40 s. The final extension was 72°C for 1 min. Gel electrophoresis was performed using 1% TBE agarose gels with ethidium bromide (25 min at 100V, Horizontal Mini Gel Electrophoresis System, Fisher Scientific, Massachusetts, USA). PCR results were imaged with a Syngene GeneFlash Gel Imaging System (MD, USA).

Image quantification

Relative band brightness was quantified using ImageJ (National Institutes of Health, MD, USA). For each band, two 18 × 20 pixel regions of interest (ROI) were defined around the band itself and around the lane background immediately below it. Relative band brightness was calculated as a ratio of the band ROI mean intensity over the background ROI mean intensity.

Data analysis

The data were analyzed using Python and Jupyter Notebook. Statistical analyses were performed using the Scipy package (https://scipy.org/).

Figure 1. Hold technique for rectal swab collection.

(A) Dorsal view demonstrating hold for young or small rats (<200 g). (B) Ventral view of hold for young or small rats. (C) Positioning inoculating loop for collection in small male rats. (D) Positioning inoculating loop for collection in small female rats. (E) Hold position for large rats (>200 g). (F) Close-up view of loop insertion. (G) Scraping technique.

Results

Scrape number

Rectal swabbing requires gently scraping the rectum lining (Figure 1), but the number of scrapes needed per sample was not specified in the original report [13]. Institutional guides recommend different ranges of scrapes be made [16], but it is unknown if or how these values have been evaluated for efficacy. This experiment assessed how the number of scrapes affects the quality of the sample's PCR amplification outcome. Given that longer PCR products can be challenging to amplify with crude DNA preparations, the effectiveness of the procedure was evaluated on two products of differing length: Sox21 (273 bp), and Pvalb^Cre (1803 bp).

It was anticipated that samples with more scrapes would create brighter bands on PCR, but results showed otherwise. Instead, samples from 2 to 20 scrapes all yielded enough DNA to produce visible and comparably bright bands (Figure 2A). The scrape number was not correlated with the brightness of the Pvalb^Cre PCR product (Figure 2B; r² = 0.0067; p = 0.50) or the Sox21 product (Figure 2C; r² = 0.0019; p = 0.72). This suggests that any number of scrapes from 2 to 20 can collect sufficient DNA for PCR amplification. Thus, we recommend that at least 2–8 scrapes be collected per sample.

Figure 2. PCR outcome does not depend on number of scrapes collected per sample.

(A) Example PCR gel image with samples of varying scrape number collected from three rats of different ages. (B) Scatter plot of Pvalb^Cre PCR product band brightness versus rectal scrape number (n.s. r² = 0.0067; p = 0.50). (C) Scatter plot of Sox21 PCR product band brightness versus rectal scrape number (n.s. r² = 0.0019; p = 0.72).

Sample number

The original report collected one sample per animal [13], but did not specify whether collecting more samples would ensure more reliable results. To determine the optimal number of samples to take, it was important to evaluate the consistency of Pvalb^Cre and Sox21 product amplification. PCR was performed on four rectal swab samples from each known-positive animal previously used to assess scrape number (n = 12). Both Pvalb^Cre (1803 bp) and Sox21 (273 bp) product bands were observed for most PCRs, however, some reactions only produced the Sox21 band and not the Pvalb^Cre band. Given that the Pvalb^Cre product is longer than the Sox21 product, it may be more difficult to amplify due to potential degradation of longer strands in the crude DNA mixture. Therefore, while the rectal swab technique reliably collected sufficient DNA for amplification, different length target sequences may vary in amplification success during PCR.

Due to this variability, the number of samples needed from each animal to maximize the likelihood of detecting a true-positive PCR result was assessed. Statistical sampling was used to simulate the effect of taking 1–4 samples from each of the 12 positive rats on the likelihood of observing a positive PCR outcome. For instance, a collection was simulated with two samples per rat by randomly selecting two of their four experimentally observed outcomes. After doing this for all 12 rats, the detection rate was defined as the proportion of rats that had at least one positive result. This simulation was then repeated 10,000 times and the average detection rate was calculated.

These simulations indicate that one rectal swab sample is sufficient to produce a visible PCR band 75% and 94% of the time for Pvalb^Cre and Sox21, respectively (Figure 3). However, taking two samples per animal increased this success rate to 89% for Pvalb^Cre and 97% for Sox21. The largest increase in amplification success was seen between one and two samples (14.52% for Pvalb^Cre and 3.55% for Sox21) followed by marginal improvement with every additional sample taken (2nd–3rd: 5.98% and 3rd–4th: 2.34% for Pvalb^Cre; 2nd–3rd: 1.48% and 3rd–4th: 0.63% for Sox21). As such, we recommend taking two samples per rat to maximize the probability of detecting a true-positive PCR result with the least additional effort.

Figure 3. Taking 2 samples per rat increases the probability of detecting a true-positive PCR result.

(A) Bar plot of probability of observing at least one visible Pvalb^Cre PCR product band given the number of samples collected from a known-positive Pvalb^Cre rat. (B) Bar plot of probability of observing at least one visible Sox21 PCR product band given the number of samples collected.

Fecal matter & cellular debris

Two potential sources of contamination during the rectal swab procedure are fecal matter from sample collection and cell debris from DNA extraction. Rectal swabbing inherently risks small amounts of fecal matter contamination, and PCR guidelines typically specify that cell debris must be separated from DNA supernatant. For these reasons, it was important to assess whether collection should be repeated in cases where visible fecal matter or cell debris remains in the sample.

To do this, the effect of fecal matter and cell debris on PCR success was measured (Figure 4A). The presence of fecal matter did not significantly affect the brightness of the resulting Pvalb^Cre band (Figure 4B; p = 0.31) or Sox21 band (Figure 4C; p = 1.0) on PCR. As fecal matter did not affect PCR outcome quality, small amounts of feces on the collection loop do not warrant repeating collection. Additionally, uncentrifuged cell debris did not significantly affect the resulting Pvalb^Cre band brightness (Figure 4D; p = 0.22) or Sox21 band brightness (Figure 4E; p = 0.84). Thus, incompletely centrifuged cell debris does not require sample recollection either.

Figure 4. Fecal matter and cell debris do not affect PCR amplification outcomes.

(A) Sample PCR including proper rectal swab samples, samples with visible fecal matter and samples with persistent cell debris from 6 rats of different sexes and ages (W: weeks). (B) Scatter plot of Pvalb^Cre band brightness on PCR versus fecal matter presence (n.s. p = 0.31). (C) Scatter plot of Sox21 band brightness on PCR versus fecal matter presence (n.s. p = 1.0). (D) Scatter plot of Pvalb^Cre band brightness on PCR versus cell debris presence (n.s. p = 0.22). (E) Scatter plot of Sox21 band brightness on PCR versus cell debris presence (n.s. p = 0.84). Statistical testing was conducted using the Wilcoxon signed-rank test.

Ear clip genotyping

Rectal swabbing was compared to the common yet invasive ear clipping technique (Figure 5). Ear clipping produced no significant difference in Pvalb^Cre band brightness (Figure 5B; p = 0.094) but produced significantly brighter Sox21 bands (Figure 5C; p = 0.031). Despite differences in band brightness, however, both techniques produced visible bands that clearly convey the genotype of a transgenic animal (Figure 5A). Thus, rectal swabbing can produce DNA of sufficient quality for use in PCR reactions, comparable in utility to the established method of ear clipping.

Figure 5. Ear clipping produced brighter Sox21 bands than rectal swabbing, but both techniques produced Pvalb^Cre bands of statistically comparable brightness.

(A) Sample gel including PCR-amplified rectal swab and ear clip samples from 6 rats of different sexes and ages (W: weeks). (B) Paired scatter plot of Pvalb^Cre band brightness on PCR versus DNA collection technique (n.s. p = 0.094). (C) Paired scatter plot of Sox21 band brightness on PCR versus DNA collection technique (p = 0.031). Statistical testing was conducted using the Wilcoxon signed-rank test.

Oral swab genotyping

Rectal swabbing was also compared to another minimally invasive collection technique, oral swabbing (Figure 6A). Rectal swabbing produced significantly brighter Pvalb^Cre bands (Figure 6B; p = 0.0039) and Sox21 bands (Figure 6C; p = 0.0039) than oral swabbing. This aligns with previous reports that oral mucosa cells have a lower expected DNA yield than other techniques [5]. Additionally, oral swab collection posed several technical challenges. It was difficult to insert the collection loop into the rat's mouth and scrape the buccal cavity without the rat either chewing off the collection loop, grabbing the loop with its forepaws or expelling the loop with its tongue. Restraining the animal for long enough to access the mouth is difficult because it requires the experimenter to use one hand to hold the animal around its front legs while simultaneously using the other hand to insert the loop. This is especially challenging for large rats or rats that are unaccustomed to handling, as they are more difficult to secure and tend to struggle. These results show that rectal swabs produced more reliable genotyping results compared with oral swabs (Figure 6).

Figure 6. Rectal swabs consistently produced brighter Sox21 and Pvalb^Cre bands on PCR.

(A) Sample gel including PCR-amplified rectal swab and oral swab samples from 9 rats of different sexes and ages (W: weeks). (B) Paired scatter plot of Pvalb^Cre band brightness on PCR versus DNA collection technique (p = 0.0039). (C) Paired scatter plot of Sox21 band brightness on PCR versus DNA collection technique (p = 0.0039). Statistical testing was conducted using the Wilcoxon signed-rank test.

Discussion

This report's findings informed the creation of a protocol for rectal swab collection and genotyping (available as Supplemental data). This complete process of rectal swab genotyping, including sample collection, PCR and gel electrophoresis, takes approximately 3–5 h for a typical litter of laboratory rats with 8–16 pups. The previous protocol outlined by Lahm et al. used overnight DNA extraction [13], whereas this protocol requires approximately 5–10 min for DNA extraction for one sample. One report claims that rectal swab genotyping requires more reagents than existing methods [17], however, our protocol uses the same set of reagents and manufacturer guidelines designed to process tissue samples. The use of these commercial kits also demonstrates that rectal swab samples can be processed at the same speed and cost per reaction as invasively collected tissue samples. However, a trade-off for fast processing is purity. Quantifying the concentration or purity of the crude DNA sample poses challenges because the crude mixture will likely contain compounds that interfere with spectrophotometric methods for quantification. While these crude samples are sufficient for quick end-point PCR genotyping, they may be insufficient for real-time PCR, qPCR or Southern blotting. This is a limitation of genotyping with the current protocol.

The rectal swab technique is also advantageous for animal welfare. It is a minimally invasive technique that does not require amputation like more popular invasive methods. Further, physiological measurements indicate that rectal swabs do not cause long-term distress to animals [12]. For these reasons, rectal swabbing can be repeated many times without injuring the rat or running out of tissue to collect, which is possible for many invasive techniques. Tail clipping, for instance, can only be completed twice, with the second sample being highly discouraged [8,9]. We also tested oral swabbing, an alternative minimally invasive technique, but this did not reliably produce amplified products, similar to previous findings [5]. Additionally, we note that spatially unique patterns from ear clipping are commonly used to identify individual animals that share a single cage. Because rectal swabbing only serves to collect DNA, alternative methods are necessary to mark animals for identification. Another reported concern is bleeding during rectal swabbing in mice [12,16], which implies injury and thus raises the question of invasiveness. However, we have not observed any bleeding with adult or weaning-aged rats while using our protocol, demonstrating that rectal swabbing can be conducted safely and effectively without causing injury.

Conclusion

These results indicate that the rectal swab technique can effectively collect sufficient genomic DNA for PCR amplification. The number of scrapes made along the rectal epithelium did not affect PCR outcome quality, allowing us to recommend a lower range of 2–8 scrapes per sample. Taking two samples per rodent further increased the likelihood of PCR success. Additionally, fecal matter and cell debris did not affect PCR outcome quality, indicating that collection need not be repeated in these cases. Rectal swabbing demonstrated similar utility as ear clipping, also collecting sufficient DNA to produce visible PCR bands for sequences up to ∼1800 bp. When compared to oral swabbing, rectal swabbing consistently produced clearer genotyping results. Finally, our protocol leverages the efficiency of commercial direct-from-tissue PCR reagents. This enables DNA extraction and PCR with a time frame and cost that are comparable to what invasively collected tissue samples require. We conclude that rectal swabbing is similarly effective as ear clipping for genotyping rodents with end-point PCR, while remaining minimally invasive, repeatable and comparably quick and inexpensive.

Future perspective

Minimally invasive DNA collection is important for reducing distress to animals from injury or extremity amputation. This is especially important for studies investigating locomotion and fine limb movement. Additionally, fast and convenient genotyping methods will be highly suitable for laboratories with minimal molecular biology equipment. Our protocol can potentially be applied to laboratory species other than rats and mice, including gerbils, hamsters and guinea pigs. PCR genotyping is used in these model species to determine natural genetic variation or for sexing. Further, rectal swabbing may be used to collect DNA from small animals in field studies where fecal samples are difficult to collect, or where tissue sampling through blood or appendage amputation poses an even higher risk of harm. Our protocol thus provides researchers with a convenient method for DNA collection with a wide range of applications in and out of the laboratory.

Author contributions

AE Kaye, JW Proctor-Bonbright and JY Yu conceived the project. AE Kaye conducted the experiments. AE Kaye developed the protocol with input from JW Proctor-Bonbright. AE Kaye and JY Yu analyzed the data and wrote the manuscript with input from JW Proctor-Bonbright.

Financial disclosure

This work was supported by a Whitehall Foundation research grant (2022-12-017, JY Yu) and the University of Chicago Neuroscience Early Stage Scientist Training Program from the National Institute of Neurological Disorders and Stroke (5R25NS117360, AE Kaye and JW Proctor-Bonbright). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that all procedures were performed with approval from the Institutional Animal Care and Use Committee at the University of Chicago, according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, and have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.2144/btn-2024-0023

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Additional information

Funding

This work was supported by a Whitehall Foundation research grant (2022-12-017, JY Yu) and the University of Chicago Neuroscience Early Stage Scientist Training Program from the National Institute of Neurological Disorders and Stroke (5R25NS117360, AE Kaye and JW Proctor-Bonbright).

Notes

1 This article has been corrected with minor changes. These changes do not impact the academic content of the article.