We would like to acknowledge NHMRC Fellowships APP1109749 and APP1111216, University of Queensland Centennial Scholarship and International Postgraduate Research Scholarship.

The study was approved by Metro South Human Research Ethics Committee and the University of Queensland Human Research&#160;Ethics Committee (HREC-13-QPAH-551 and UQ2013001551) and the University of South Australia Human Ethics Committee (200607).
1. Absorbent Microbiopsy Fabrication
<ol>
	<li>Laser cut microneedle parts from 50 &#181;m medical-grade stainless steel sheet and absorbent layer (filter paper), respectively (Figure 1). 
	Note: Other parts, including the plunger and the case, of the device are fabricated with rapid-prototyping techniques, such as 3D printing.</li>
	<li>Soak all the parts, except for the absorbent layer, in 70% ethanol (EtOH) for 5 min and air dry them for 30 min afterwards.</li>
	<li>Assemble the three-layer microneedle, with the absorbent layer in the middle (Figure 1a).</li>
	<li>Insert the assembled microneedle into the slit of plunger.</li>
	<li>Put on the spring and insert the plunger into the case (Figure 1c). Avoid the microneedle touching anything in the assembling procedure to ensure the needle quality.</li>
	<li>Seal the assembled absorbent microbiopsy in a package for &#947;-radiation sterilization before using.</li>
</ol>
2. Sampling with the Absorbent Microbiopsy
<ol>
	<li>Wear disposable gloves, and spray hands and tools, such as forceps, with 70% EtOH.</li>
	<li>Sanitize the application site with an alcohol wipe.</li>
	<li>Remove the microbiopsy from the &#947;-radiation sterilized package without touching the microneedle in the process.</li>
	<li>Load the device by pulling the plunger to compress the spring until there is a &#8216;click&#8217; sound and the plunger locks in place.</li>
	<li>Aim the device at the skin to be sampled, ensuring that the sampling area is in a fixed position.</li>
	<li>Ensure that the device is approximately perpendicular to the skin, then apply &#8805;1 kg of force to the device onto the skin. 
	Note: Applying force downwards into the skin is required to ensure sufficient penetration of the microneedle.</li>
	<li>Press the trigger and hold the device in place for a minimum of 10 s for the blood sample to be absorbed. 
	Note: If the device is released before 10 s, there is less sample captured.</li>
</ol>
3. RNA Extraction
Note: The RNA extraction procedures were modified from manufacturers&#8217; protocol for optimal isolation of RNA from microbiopsied sample. All the reagents and column used in RNA extraction are included in the kit unless indicated otherwise.
<ol>
	<li>Pull out the plunger and absorbent microneedle from the case by hands gently. Ensure that the tip of the microneedle does not come into contact with anything during removal.</li>
	<li>Remove the absorbent microneedle from the plunger. Hold onto the two holes on the microneedle with a pair of sterile forceps and pull it out.</li>
	<li>Put the intact microneedle in a 2 mL microcentrifuge tube (not from the RNA isolation kit; see Table of Materials) prefilled with 50 &#181;L of Extraction Buffer, with the needle side facing down towards the bottom. To minimize sample loss, leave the tube on dry ice for 5 min before processing the sample.</li>
	<li>Vortex briefly (3 - 5 s) to remove some of the sampled materials from the tip.</li>
	<li>Incubate the microneedle in the tube on a rocker for 30 min at 42 &#176;C. 
	Note: The buffer solution will be absorbed into the absorbent layer immediately.</li>
	<li>Place the top part of the microneedle (the opposite side from the needle) level with the top edge of the 2 mL microfuge tube using sterile forceps.</li>
	<li>Close the lid of the tube such that the top of the microneedle is held in place between the cap and tube.</li>
	<li>Spin the tube at 16,000 x g or at maximum speed for 30 s to remove the sampled materials from the absorbent layer of the device.</li>
	<li>Remove the microneedle carefully with forceps without dipping back into the buffer solution. Keep the tube and discard the microneedle.</li>
	<li>Centrifuge the samples at 3,000 x g for 2 min. Pipette the supernatant containing the extracted RNA carefully into a new microcentrifuge tube.</li>
	<li>Add 250 &#181;L of Conditioning Buffer onto the purification column filter membrane and incubate for 5 min at room temperature.</li>
	<li>Centrifuge the column in the collection tube at 16,000 x g or at maximum speed for 1 min.</li>
	<li>Add 50 &#181;L of 70% EtOH into the collected sample from Step 3.9. Mix well by gently pipetting up and down.</li>
	<li>Transfer the mixture (~100 &#181;L) into the preconditioned purification column.</li>
	<li>Centrifuge for 2 min at 100 x g immediately, followed by a centrifugation at 16,000 x g for 30 s to remove the flow-through.</li>
	<li>Add 100 &#181;L of Wash Buffer 1 (W1) into the purification column and centrifuge for 1 min at 8,000 x g.</li>
	<li>Prepare DNase solution by adding 5 &#181;L of DNase I Stock Solution to 35 &#181;L of Buffer RDD in a separate microcentrifuge tube for each sample. Mix by gently inverting. 
	Note: The DNase treatment is to be performed on the purification column, instead of in the PCR plate afterwards, because of the quantity of the sample.</li>
	<li>Add 40 &#181;L of DNase incubation mix directly into the purification column membrane. Incubate at room temperature for 15 min.</li>
	<li>Add 40 &#181;L of W1 into the purification column membrane. Centrifuge at 8,000 x g for 15 s.</li>
	<li>Pipette 100 &#181;L of Wash Buffer 2 (W2) into the purification column after the DNase treatment and centrifuge for 1 min at 8,000 x g.</li>
	<li>Add another 100 &#181;L of W2 into the purification column and centrifuge for 2 min at 16,000 x g. Check the purification column for any residual wash buffer. Re-centrifuge at 16,000 x g for 1 min if any wash buffer remains.</li>
	<li>Transfer the purification column to a new 0.5 mL microcentrifuge tube provided in the kit.</li>
	<li>Pipette 11 &#181;L of RNase-free water (not provided in the RNA isolation kit), instead of Elution Buffer (EB), directly onto the membrane of the purification column. Gently touch the tip of the pipette to the surface of the membrane while dispensing the RNase-free water to ensure maximum absorption of RNase-free water into the membrane. Incubate at room temperature for 2 min.</li>
	<li>Centrifuge the column for 1 min at 1,000 x g to distribute the RNase-free water in the column, then centrifuge for 16,000 x g at 1 min to elute RNA. 
	Note: RNA quantification is not recommended due to the small quantity of the sample.</li>
	<li>Stop point #1: Store the total RNA sample at -80 &#176;C if cDNA synthesis is not performed immediately. 
	Note: Avoid freezing if not necessary given the low concentration of RNA sample.</li>
</ol>
4. cDNA Synthesis
<ol>
	<li>Thaw the frozen sample on ice (from Step 3.25).</li>
	<li>Synthesize cDNA from total RNA with cDNA synthesis kit according to the procedure below.</li>
	<li>Prepare the reaction mix: 11 &#181;L of total RNA, 4 &#181;L of 5x TransAmp Buffer, 1 &#181;L of reverse transcriptase, 4 &#181;L of DNase/RNase-free water. Mix gently by pipetting.</li>
	<li>Run reverse transcription on a thermal cycler apparatus: 25 &#176;C for 10 min, 42 &#176;C for 15 min, followed by a final reverse transcriptase inactivation step at 85 &#176;C for 5 min.</li>
	<li>Stop point #2: Store the reverse transcribed sample at -80 &#176;C until use.</li>
</ol>
5. qPCR Reaction and Data Analysis
Note: The primers for qPCR reactions were designed to span intron boundaries to avoid the amplification of genomic DNA using NCBI Primer BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/). The reference gene used in this study is RPLP0 (see the Discussion section for more information).
<ol>
	<li>Thaw the frozen sample on ice (from Step 4.5).</li>
	<li>Perform qPCR with the qPCR master mix kit according to the procedure below.</li>
	<li>Prepare master mix containing the followings per reaction: 5 &#181;L of 2x qPCR reaction mix, 0.4 &#181;L of 10 &#181;M forward primer, 0.4 &#181;L of 10 &#181;M reverse primer, 2.2 &#181;L of DNase-free water. Mix gently by pipetting. Set up the samples in triplicates.</li>
	<li>Pipette 8 &#181;L of the master mix into each well of a 384-well PCR plate first and then 2 &#181;L of cDNA from each sample (a total of 10 &#181;L per well). 
	Note: No template control (NTC) and no reverse transcriptase control (-RT) are included as negative controls.</li>
	<li>Seal the plate with adhesive film, and centrifuge briefly.</li>
	<li>Run the amplification reaction on a real-time thermal cycler: 95 &#176;C for 2 min (polymerase activation), followed by 40 cycles of 95 &#176;C for 5 s (denaturation), 60 &#176;C for 10 s (annealing) and 72 &#176;C for 10 s (extension).</li>
	<li>Calculate the RNA concentration of samples by interpolating from a standard curve prepared from templates with known concentrations.
	<ol>
		<li>Create a New Experiment in the software.</li>
		<li>Click Define and Set Up Standards to set up serial dilution for plates.</li>
		<li>Select and arrange the wells for standards and samples. Click Apply.</li>
		<li>Click Analyze in the Result tab to assess the standard curve. Confirm the slope, R2 value, amplification efficiency and error meet the experimental criteria.</li>
		<li>Check the quantities of the unknown samples visually on the standard curve based on their Ct values. 
		Note: The construction of standard curve, including the choice of dilution factor, is performed according to published protocols11,12,13.</li>
	</ol>
	</li>
	<li>Perform gene expression analysis of mRNA by using the &#916;Ct method (Ct is normalized to a refence gene).
	<ol>
		<li>Subtract the Ct value of target gene from the Ct value of reference gene to obtain the &#916;Ct value.</li>
		<li>Average the &#916;Ct values of technical replicates.</li>
		<li>Plot and analyze the gene expression with statistics software11,14 (see Table of Materials). 
		Note: Alternatively, the gene expression analysis is performed according to published protocols13.</li>
	</ol>
	</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The skin microbiopsy. , (2015).</li><li>Dhingra, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+contact+dermatitis+skin+identifies+allergen-dependent+differences+in+immune+response.">Molecular profiling of contact dermatitis skin identifies allergen-dependent differences in immune response.</a> Journal of Allergy and Clinical Immunology. 134, 362-372 (2014).</li><li>Chapman, P. 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GraphPad - FAQ 1753 - Prism 3 -- Calculating &amp;#34;Unknown&amp;#34; Concentrations using a Standard Curve Available from: <a target="_blank" href="https://www.graphpad.com/support/faq/prism-3-calculating-unknown-concentrations-using-a-standard-curve/">https://www.graphpad.com/support/faq/prism-3-calculating-unknown-concentrations-using-a-standard-curve/</a> (2018)</li><li>Kubista, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Molecular Aspects of Medicine. 27, 95-125 (2006).</li><li>Goni, R., Garc&#237;a, P., Foissac, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The qPCR data statistical analysis. , 1-9 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GraphPad Statistics Guide. , (1995).</li><li>Goldstein, N. 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No conflicts of interest declared.

These results demonstrate that the absorbent microbiopsy can be used as a tool for simple and minimally invasive sampling of skin and blood mixture for molecular characterization. Device application in accordance with our protocol is essential for obtaining reliable results as shown by the difference in RNA amount recovered with different application protocols (Figure 3). Once the sample is extracted, the subsequent sample processing step for RNA extraction is highly similar to established protocols15,16. Besides from the initial steps in RNA extraction that are modified for the absorbent microbiopsy, another key change is the use of RNase-free water to improve the results for downstream applications. Moreover, it is worth mentioning that the reference gene used in this study is RPLP0. RPLP0, whose function is well known for different cell and tissue types17, has been reported as a suitable reference gene for use in non-melanoma skin cancers and precancerous lesions18.
One of the main limitations of the device is the removal of the microbiopsy from the device, which is time-consuming and potentially increases the chance of sample loss, especially for sensitive samples like RNA. Nevertheless, the issue can be overcome by pre-cooling all the sterile processing tools, such as the 2 mL microcentrifuge tubes, on dry ice.
The use of the absorbent microbiopsy is simple and does not require intensive training. Conventional biopsy is not necessary as microbiopsy permits molecular characterization with established molecular diagnosis techniques, such as RT-qPCR. To further quantify and demonstrate the sampling ability of absorbent microbiopsy, previous literature that involved RNA extraction from human skin tissue was investigated. From a typical 3.0 or 4.0 mm skin punch biopsy, three studies have reported the extracted RNA amounts ranged from 50 to 200 ng per mg of skin tissue19,20,21. Comparing with the 1.43 ng of RNA that was recovered from the absorbent microbiopsy on average (Figure 3), the weight of sampled skin tissues is expected to range from 0.29-115 &#181;g based on the same RNA-to-tissue ratio from skin punch biopsy studies.
This protocol is not without potential pitfalls, though some of the problems can be easily overcome. For example, the RNA extraction data suggested a considerable&#160;variation even with the 10-s holding time (Figure 3). To address the problem, one potential solution involves optimizing the application protocol. Parameters such as force applied to the skin and holding time should be tested and optimized to reduce the variations in sample extraction. The other potential issue is the removal of microbiopsy from the device, which might impact the sample integrity and recovery. Although removing the microbiopsy for RNA extraction is an effective approach, the entire procedure is tedious, and the sample might be exposed to contamination in the process. Therefore, the modification of the sample processing protocol is highly desired in order to ensure sample integrity and prevent sample loss.
Once the above two issues are addressed, it is expected that the device will become a standard tool for clinical research. It is important to note that the device captures both skin and blood sample simultaneously and this should be taken into consideration when analyzing gene expression data. In conclusion, this protocol describes the details of performing absorbent microbiopsy as an easy and minimally invasive tool for combined skin and blood sampling and the subsequent sample processing for relative gene expression profiling.

Skin biopsy is one of the most essential techniques in dermatology for skin sampling and subsequent diagnosis of skin diseases through histopathological assessment. The biopsy technique involves a medical professional using a blade or punch biopsy to remove the suspicious lesion on patient&#39;s skin for examination1. Though the technique is effective, it is highly invasive and limits clinical research as the end point usually involves molecular biology techniques2,3. Molecular analysis of skin diseases has the potential to provide highly specific biological information that histopathological analysis cannot, thus facilitating&#160;drug discovery and disease diagnosis4,5. Besides, the sample demand in most molecular techniques is comparatively small and may lead to a reduction in animal use and permit a larger number of replicates. Therefore, there is a clear need for an alternative technique that enables molecular analysis in clinical research and lowers risk for participants.
To address such a need in the field, our group has developed a novel microneedle-based diagnostic platform, absorbent microbiopsy, that enables the collection of a tiny amount of skin mixed with blood in a simple and minimally invasive manner6. The purpose of this publication is to describe the absorbent microbiopsy as a sampling tool to facilitate molecular analysis through RNA extraction in clinical research.
Previously, we have described the first version of microbiopsy, skin microbiopsy, which consists of a microneedle made of a three-layer steel plate design to extract tiny pieces of skin tissues7. The novelty of this device comes from the multiple contact points from the microneedle that permits efficient tissue extraction3. In contrast, a circular skin punch biopsy provides only one contact point and simply tears the skin without capturing any sample in some cases. Based on the skin microbiopsy, we recently developed the absorbent microbiopsy which has both blood and skin sampling capabilities. The device has been shown to be feasible for use in resource-poor areas in a recent epidemiological study6.
Due to its simple design, the absorbent microbiopsy can be performed within a few seconds and does not require extensive training. Additionally, local anesthetic is not needed, and the application site does not cause scarring. The present protocol enables researchers or medical professionals without relevant sampling training to obtain targeted skin data for molecular analysis. We expect microsampling devices to become routine in skin research in the future.
Although the microbiopsy has been reported in other skin disease studies that involved molecular diagnostic techniques6,8,9,10, such as human papilloma virus DNA detection, this protocol is the first to demonstrate the details of the sample extraction and processing techniques for the absorbent microbiopsy. Further, this is the first report describing the relative gene expression profiling of skin and blood cells in microbiopsy samples.

<table><tbody><tr><td>&lt;strong&gt;Absorbent microbiopsy fabrication and sampling&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Absorbent Microbiopsy</td><td>Trajan Scientific and Medical</td><td>N/A</td><td>https://www.trajanscimed.com/</td></tr><tr><td>Whatman filter paper, Grade 1</td><td>Sigma Aldrich</td><td>WHA1001325</td><td>N/A</td></tr><tr><td>&lt;strong&gt;RNA extraction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PicoPure RNA Isolation Kit</td><td>ThermoFisher</td><td>KIT0214</td><td>Including all buffer soltuions described in protocol</td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>ThermoFisher</td><td>10977015</td><td>Improving RNA quality in RNA elution step</td></tr><tr><td>2.0 mL Microcentrifuge Tube, Sterile</td><td>Thomas Scientific</td><td>1226S74</td><td>N/A</td></tr><tr><td>Microcentrifuge 5415R</td><td>Eppendorf</td><td>Z605212</td><td>N/A</td></tr><tr><td>&lt;strong&gt;cDNA synthesis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SensiFAST cDNA Synthesis Kit</td><td>Bioline</td><td>BIO-65053</td><td>N/A</td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System</td><td>Bio-Rad</td><td>1855196</td><td>N/A</td></tr><tr><td>&lt;strong&gt;qPCR reaction and data analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SensiFAST SYBR Lo-ROX Kit</td><td>Bioline</td><td>BIO-94005</td><td>Including reagents described in qPCR reaction steps</td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>ThermoFisher Scinentific</td><td>4311971</td><td>N/A</td></tr><tr><td>MicroAmp Optical 384-Well Reaction Plate</td><td>ThermoFisher Scinentific</td><td>4343370</td><td>N/A</td></tr><tr><td>QuantStudio 6 Flex Real-Time PCR System</td><td>ThermoFisher Scinentific</td><td>4485694</td><td>N/A</td></tr><tr><td>GraphPad Prism (v6.04)</td><td>GraphPad</td><td>N/A; Windows version</td><td>Plotting and statistical analysis in qPCR data analysis steps</td></tr><tr><td>&lt;strong&gt;PCR primers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;RPLP0&lt;/em&gt; F</td><td>Sigma Aldrich</td><td>N/A</td><td>ATC AAC GGG TAC AAA CGA GTC</td></tr><tr><td>&lt;em&gt;RPLP0&lt;/em&gt; R</td><td>Sigma Aldrich</td><td>N/A</td><td>CAG ATG GAT CAG CCA AGA AGG</td></tr><tr><td>&lt;em&gt;TYR&lt;/em&gt; F</td><td>Sigma Aldrich</td><td>N/A</td><td>TCA GCA CCC CAC AAA TCC TAA</td></tr><tr><td>&lt;em&gt;TYR&lt;/em&gt; R</td><td>Sigma Aldrich</td><td>N/A</td><td>AAT CGG CTA CAG ACA ATC TGC</td></tr><tr><td>&lt;em&gt;KRT14 &lt;/em&gt;F</td><td>Sigma Aldrich</td><td>N/A</td><td>CCT CCT CCC AGT TCT CCT</td></tr><tr><td>&lt;em&gt;KRT14 &lt;/em&gt;R</td><td>Sigma Aldrich</td><td>N/A</td><td>ACA CCA CCT TGC CAT CG</td></tr><tr><td>&lt;em&gt;CD3E &lt;/em&gt;F</td><td>Sigma Aldrich</td><td>N/A</td><td>CAA AGG GGA CAA AAC AAG GAG</td></tr><tr><td>&lt;em&gt;CD3E &lt;/em&gt;R</td><td>Sigma Aldrich</td><td>N/A</td><td>GTT CTC CAG AGG GTC AGA TG</td></tr><tr><td>&lt;em&gt;CD19 &lt;/em&gt;F</td><td>Sigma Aldrich</td><td>N/A</td><td>TTC TGC CTG TGT TCC CTT G</td></tr><tr><td>&lt;em&gt;CD19 &lt;/em&gt;R</td><td>Sigma Aldrich</td><td>N/A</td><td>GCG TCA CTT TGA AGA ATC TCC T</td></tr></tbody></table>

absorbent microbiopsy sampling and rna extraction for minimally invasive, simultaneous blood and skin analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

We have previously reported that the microneedle of the skin microbiopsy penetrates the skin approximately 500 &#181;m deep7. The microneedle design of skin microbiopsy is highly similar to the one on absorbent microbiopsy (Figure 2a). While the absorbent microbiopsy consisted of an absorbent layer in the middle for blood absorption, the skin microbiopsy contained a channel for mechanical capturing of skin tissues. The use of absorbent layer also led to a slight difference in the microneedle dimension (absorbent: 1.50 x 0.50 x 0.21 mm vs skin: 1.50 x 0.50 x 0.15 mm).
Figure 2b shows the application sites 5 min after absorbent and skin microbiopsies were applied on the left volar arm of male volunteer. Due to the similarity between the two microneedle designs, the application sites from both devices were comparable, with minor erythema. Both application sites were not noticeable after 48 h with no scars left behind. This supports the hypothesis that this minimally invasive device has the potential to help screen multiple application sites or to be performed on a regular basis.
Figure 2c shows a representative picture of the absorbent layer of the absorbent microbiopsy after being applied to the volar arm of a male volunteer. As shown in the figure, a few tiny pieces of skin were captured near the tip of the microneedle, and some blood was absorbed into the filter paper. This indicates that the device penetrated the skin and captured both skin and blood simultaneously within the same absorbent microbiopsy matrix. Figure 2d shows a post-sampling skin microbiopsy, the previous generation of microbiopsy, for comparison. The channel of the skin microbiopsy captured a small piece of skin, but the amount of blood on the microneedle was small relative to the absorbent microbiopsy. Both application sites were not noticeable within 48 h. In the experiment, the absorbent microbiopsy was applied with a 10-s holding time post-application, while the skin microbiopsy was immediately released after applying due to the difference in design.
As shown in Figure 3a, two groups of absorbent microbiopsy were involved in this experiment based on the application protocol: &#39;Immediate release&#39; and &#39;10-s holding&#39;. For the &#39;Immediate release&#39; group, the device was applied using the same original skin microbiopsy protocol, with the device being removed from the application site immediately after application. For the &#39;10-s holding&#39; group, the device was held in place after the application for 10 s to improve sample collection. The two groups of absorbent microbiopsy were set up to demonstrate how the application approach could affect the sample amount. The 10-s holding time was chosen as a clinically reasonable time to demonstrate that the application time affects the amount of recoverable sample.
The amount of RNA recovered from the two absorbent microbiopsy groups were 0.33 &#177; 0.39 ng for &#39;Immediate release&#39; and 1.43 &#177; 0.88 ng for &#39;10-s holding&#39; (Figure 3a, n = 20), suggesting a 4-fold increase with the extra holding time. This indicates that applying the absorbent device with the 10-s holding time resulted in more RNA extracted. The difference may be due to the presence of increased blood sample in the absorbent layer (Figure 3c). Indeed, the &#39;Immediate release&#39; group (Figure 3b) failed to collect a similar amount of blood with the absorbent layer as compared to the &#39;10-s holding&#39; group (Figure 3c). It should also be noted that most immediately released microbiopsies were close to the x-axis, suggesting they were negative events or displayed a very low amount of RNA. Therefore, the result validated the hypothesis that the holding time would have an impact on the performance of the device as it might take time for the blood to be absorbed by the absorbent layer.
Since the device was designed for blood and skin sampling, qPCR was used to detect the expression of skin and blood biomarkers for both devices for comparison. We used tyrosinase, TYR, as a biomarker for melanocytes and KRT14 as a biomarker for keratinocytes in the viable epidermis. Skin microbiopsy samples were included in the experiment for comparison. As shown in Figure 4, though skin and absorbent microbiopsies were applied differently due to the difference in design, the expression levels for both skin biomarkers TYR and KRT14 were comparable for both devices as indicated by the data. White blood cell biomarkers (CD3E, T cells and CD19, B cells) were found to be more prevalent in absorbent microbiopsy samples than in skin microbiopsy samples. This result suggests that the absorbent microbiopsy performed better in blood collection but still maintained the capacity for capturing skin as compared to the skin microbiopsy (n = 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58614/58614fig1.jpg" /> 
Figure 1. The fabrication of absorbent microbiopsy. (a) The three-layer microneedle. (b) All parts of the device. (c) The assembled absorbent microbiopsy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58614/58614fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58614/58614fig2.jpg" /> 
Figure 2. The absorbent microbiopsy was able to capture skin and blood samples simultaneously without leaving a scar on the left volar arm of male volunteer. (a) A comparison between the microneedles of absorbent and skin microbiopsies. (b) Application sites left by absorbent and skin microbiopsies 5 min after application. (c,d) Microneedles of absorbent and skin microbiopsies after application. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58614/58614fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58614/58614fig3.jpg" /> 
Figure 3. The absorbent microbiopsy captured more blood and RNA sample when applied for 10-s. (a) The 10-s post-application holding time resulted in a higher amount of RNA than releasing the device immediately (n = 20). Error bars represent S.D. from the mean (****p&#60;0.0001). (b,c) Representative pictures of the absorbent microbiopsies after application with &#39;Immediate release&#39; and &#39;10-s holding&#39; approaches. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58614/58614fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58614/58614fig4.jpg" /> 
Figure 4. The comparison of mRNA expression levels between skin and absorbent microbiopsies (n = 5). The gene expression had been normalized with reference gene RPLP0. Error bars represent S.D. from the mean (*p&#60;0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58614/58614fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis at JoVE.com

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

absorbent-microbiopsy-sampling-rna-extraction-for-minimally-invasive

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K Views.  University of South Australia. Deze methode kan de oprichting van biomarkers voor huidziekten vergemakkelijken en ook het risico voor deelnemers aan klinisch onderzoek verminderen. De belangrijkste voordelen van deze techniek zijn dat het geen intensieve training vereist en geen littekens achterlaat. Vijf minuten voor de microbiopsie, plaats een lege twee milliliter centrifuge buis op droog ijs gedurende vijf minuten.Het dragen van wegwerphandschoenen, spray de handen en gereedschappen met 70% ethanol voor het ontsm...

Video: Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Neuroscience

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Cancer Research

Establishment of a Clinic-based Biorepository

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

Cutaneous tumors are often discarded following Mohs micrographic surgery. A protocol is described here that enables clinical support staff to effectively process and store cutaneous tumor (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) samples for downstream laboratory applications without interfering with clinical operations.

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Biology

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a &#8216;blind-swabbing&#8217; approach will co-sample cellular material from the different individuals, even if the individuals&#8217; cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim&#8217;s DNA may be found in significant excess thus masking any potential perpetrator&#8217;s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, &#8216;smart analysis&#8217; method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 &#181;l microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., &#8220;clumps&#8221;) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in &#8216;touch DNA&#8217; samples, together with an enhanced amplification protocol involving a one-step 5 &#181;l micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...

Specimen Collection and Analysis of the Duodenal Microbiome

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut microbiome has been typically studied through fecal specimen collections. The ease of obtaining fecal samples has resulted in many studies that have revealed information concerning the distal luminal gastrointestinal tract. However, few studies have addressed the importance of the microbiome in the proximal gut. Given that the duodenum is a major site for digestion and absorption, its microbiome is relevant to nutrition and liver disease and warrants further investigation. Here we detail a novel method for sampling the proximal luminal and mucosal gut microbiome in human subjects undergoing upper endoscopy by obtaining duodenal aspirate and biopsies. Specimen procurement is facile and unaffected by artifacts such as patient preparatory adherence, as might be the case in obtaining colonic samples during colonoscopy. The preliminary results show that the luminal and mucosal microbiomes differ significantly, which is likely related to environmental conditions and barrier functions. Therefore, a combination of duodenal aspirate and biopsies reveal a more comprehensive picture of the microbiome in the duodenum. Biopsies are obtained from the descending and horizontal segments of the duodenum, which are anatomically close to the liver and biliary tree. This is important in studying the role of bile acid biology and the gut-liver axis in liver disease. Biopsies and aspirate can be used for 16S ribosomal RNA sequencing, metabolomics, and other similar applications.

In this manuscript, we discuss a novel method to sample and analyze the duodenal microbiome. This method provides an accurate depiction of microbial diversity and composition in the duodenum and could be useful for further investigation of the duodenal microbiome.

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

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