Hello Morjina Solaiman,

RNA extraction from skin tissue can be challenging because the hyaluronic acid-collagen matrix is difficult to homogenize. Moreover, the presence of RNases makes it more difficult.

The critical steps involved in RNA extraction from skin sample include biopsy collection, storage condition, enzymatic digestion, stator-rotor based homogenization combined with column-based RNA purification.

You may consider incorporating the following in your protocol.

1. There are two strategies to block the activity of RNases either immediate freezing after sample collection or applying stabilizing reagents at room temperature and/or lower temperature.

2. You can achieve homogenization via mincing with razor blade, use of mechanical homogenizer or the use of a motar and pestle in combination with liquid nitrogen.

The method of grinding in liquid nitrogen would be suitable for such sample. For this purpose, you need to have a large mortar and pestle which is always kept precooled in -20-degree C. The large size is necessary as it makes sure that you can work with ease, grinding the sample with force and it ensures that the tissue powder does not spill out. Make sure that there is constant cooling when you disrupt the tissue.

3. Combining collagenase and hyaluronidase digestion prior to homogenization would increase the efficiency of homogenization. If enzymatic digestion is supposed to be carried out at 37 deg C, RNase inhibitors are strongly recommended. Try using beta-mercaptoethanol as it is used to inactivate RNases. You will need to add additional amount of proteinase K for lysis.

4. Use of stator and rotor-based homogenizer is recommended to fully homogenize skin sample.

5. DNA contamination is likely to occur. So, strategies such as post-RNA extraction DNAse treatment should be considered.

6. Avoid loading the fatty layer as it may clog the column. Carry out double elution to increase RNA yield.

The below attached article may help.

https://www.nature.com/articles/s41598-019-56579-5

Best.

Hello Malcolm Nobre. Thank you so much for your response. It is really helpful. However, I need to ask do I have to use proteinase K, or I can get good yield without using it. Although i use beta-mercaptoethanol with Qiagen RLT buffer for homogenization.