How to cut RNA from paraffin-embedded human tissue wax blocks for qPCR to reduce degradation?

After FFPE RNA extraction, low concentration and short fragments are the norm, and fresh tissue standards cannot be used to judge. The recovery rate is affected by the fixed time, the retention period of the wax block, the thickness of the slice and the extraction reagent, which is generally significantly lower than that of the fresh sample. What is more important is the primer design of qPCR: the amplicon is controlled as much as possible at 70-150 bp, do not use too long products, because the RNA has been highly fragmented. Internal parameters can select genes with medium abundance, and do the dissolution curve to see if there is nonspecific amplification. If the internal parameter Ct is too large and the target gene cannot be expanded, you can increase the number of initial slices or switch to an extraction kit for FFPE and try again. Don't just look at the concentration, first confirm whether the extract can be stably amplified in the short fragment system.

After more than two years of releasing wax blocks to extract RNA, the slicing process is very critical. The thickness does not need to be too thin, 5-10 μm can be used, the amount of nucleic acid is too thin, and the dewaxing and cracking are incomplete. Pre-cooling the wax blocks before cutting can make the wax belt more complete; if the blades are replaced, clean the tool holder and tweezers with no RNase material between the different blocks to avoid cross-contamination. The exhibit is best to use DEPC to treat water or RNase-free water, do not use a regular water bath to heat it repeatedly, the temperature should not be too high, and take pieces as soon as possible after spreading. If the sliced white tablets cannot be extracted immediately, it is best to store them in a low temperature seal after drying, but do not leave them for too long. Exposure of FFPE slices to air will accelerate degradation. Each piece is first cut into about 10 pieces to try to extract, not enough to increase the amount.

Theoretically, the basic amount of RNA is not low, but there will be a lot of degradation in the wax blocks in two years, so the number of sheets cannot be calculated according to fresh tissue. It is generally recommended to cut 8-15 slices and 10 μm/slices in thickness first. If the tissue area is less than 0.5 cm ², 15-20 slices can be sliced and extracted. When extracted with FFPE-specific lysate, the digestion time of protease K can be appropriately prolonged, but it should not be overnight, and excessive digestion will not improve the complete RNA. Handle with DNase to avoid genomic contamination. The final elution volume should not be too large, 20-30 μL is more common. If the concentration is still low, priority is given to the degree of RNA fragmentation and the internal parameter Ct, rather than blindly increasing the number of pieces.

Whether xylene is used for dewaxing or an environmentally friendly dewaxing solution may not have as great an impact on the RNA yield as imagined. The key is to thoroughly dewax and whether the subsequent ethanol gradient is clean. Xylene has strong penetration and mature processes, but to operate in a fume hood, the waste liquid is treated as organic waste liquid; environmentally friendly dewaxing liquid is safe to use, but the residues of different formulations may be different. If the residues enter the lysate, it will affect the activity of protease K and RNA recovery. It is recommended that you first take a few pieces for comparison: the same wax block is cut into parallel pieces, and extracted with two dewaxing processes respectively. Run qPCR to see the internal reference Ct. Use which one is easy to use, but do not switch back and forth after fixing, otherwise the batch to batch is not comparable.