This method can facilitate establishment of bio markers for skin diseases and also reduce the risk for clinical research participants. The main advantages of this technique are that it does not require intensive training and it does not leave any scars. Five minutes before the microbiopsy, place an empty two milliliter centrifuge tube on dry ice for five minutes.
Wearing disposable gloves, spray the hands and tools with 70%ethanol before sanitizing the application site on the subject with an alcohol wipe. Remove the micro biopsy from the sterilized gamma radiation package without touching the microneedle and pull the plunger until a click is heard and the plunger locks in place. Aim the loaded device at an approximately perpendicular angle to the skin to be sampled, taking care that the sampling area is in a fixed position and apply the device onto the skin within at least one kilogram of force.
Then press the trigger and hold the device in place for at least ten seconds. If the device is released before ten seconds, there's a higher chance of variation in the blood absorption and also the subsequent analysis. When the sample has been absorbed, gently pull the plunger and absorbent microneedle from the case and use sterile forceps to pull on the two holes of the microneedle to remove it from the plunger.
Add 50 microliters of extraction buffer to the tube on dry ice and place the intact microneedle in the tube on dry ice needle side down. Be careful not to let the tip of the microneedle touch anything during the removal process. Vortex the tube for three to five seconds to collect some sampled material from the microneedle tip and incubate the tube in a heat block or water bath for 30 minutes at 42 degrees celsius.
At the end of the incubation, use sterile forceps to place the back end of the microneedle level with the top edge of the microfuge tube and close the lid of the tube such that the top of the microneedle is held in place between the cap and the tube. Centrifuge the tube to collect the sampled material from the absorbent layer of the device and use the forceps to carefully remove the microneedle without dipping the tip back into the buffer solution. Centrifuge the sample again and carefully transfer the supernatant into a new micro centrifuge tube.
Next add 250 microliters of conditioning buffer onto a purification column filter membrane for a five minute incubation at room temperature and dilute the buffer by centrifugation. Add 50 microliters of 70%ethanol to the extracted RNA sample with gentle mixing and transfer the mixture into the preconditioned purification column. Immediately centrifuge the tube two times to remove the flow-through, followed by a wash in one hundred microliters of wash buffer one.
For DNA digestion, add 40 microliters of freshly prepared DNAs incubation mix directly onto the purification column membrane for a 15 minute incubation at room temperature followed by a wash with 40 microliters of wash buffer one. At the end of the centrifugation, wash the column with 100 microliters of wash buffer two and check the purification column for any residual wash buffer. Transfer the purification column into a 0.5 milliliter micro centrifuge tube provided in the RNA extraction kit and place the tip of the pipette directly onto the membrane of the purification column to add 11 microliters of RNA sPRE water to the column.
After a two minute incubation at room temperature, centrifuge the column one time to distribute the RNA sPRE water throughout the column, and one time to elute the RNA. Then store the total RNA sample at minus 80 degrees celsius if cDNA synthesis is not performed immediately. The absorbent microbiopsy microneedle consists of two layers of steel plate with an absorbent layer in between for simultaneous sampling of the skin and blood.
The use of an absorbent microbiopsy induces only minor erythema, that is not noticeable after 48 hours. After puncture, a few tiny pieces of skin are captured near the tip of the microneedle and blood is absorbed into the filter paper. When the absorbent microneedle is removed immediately after puncture, the sample volume and subsequent amount of extracted mRNA is significantly lower than that obtained after holding for ten seconds after puncture.
Similar levels of skin marker expression are detected after the use of absorbent microbiopsy and its predecessor skin microbiopsy by quantitative PCR analysis. But a significantly higher level of white blood cell bio marker expression is detected after absorbent microbiopsy sampling, suggesting that the absorbent microbiopsy microneedle performs better for blood collection while maintaining the same capacity for skin sample capture as with skin microbiopsy. So while applying the technique, it is crucial to remember that applying the device according to what we suggest is essential for obtaining reliable results.
So following this procedure, other methods, like real-time PCR, can be followed for relatively gene expression profiling. Although we are still developing the device, we are confident that the technique has paved the way for clinical researches for performing sampling, for dermatological cosmetic or pediatric application because it's just so simple and it's minimally invasive.
