1. Low RNA extraction rate.
(1) Improperly preserved samples or samples preserved for too long. Improperly preserved samples or samples preserved for too long may lead to RNA degradation. Collect fresh samples or samples preserved in -70℃ or liquid nitrogen after liquid nitrogen flash-freezing.
(2) Inadequate homogenization or liquid nitrogen grinding. Inadequate homogenization or liquid nitrogen milling results in inadequate lysis, leading to inadequate release of RNA.
(3) Low RNA content in tissues or cells: the abundance of RNA varies in different cells and tissues, and pre-tests should be done to determine the appropriate sample starting volume.
(4) The starting amount of tissue is too little or too much: if the sample amount is too little, the RNA content in the cells and tissues is low; if the sample amount is too much, it may exceed the cleavage capacity of the lysate, and the cleavage will be incomplete, which will lead to a low rate of RNA yield or quality decline.
(5) Inadequate elution.RNase-free ddH2O was added dropwise to the center of the purification column membrane. The elution volume of the purification column is 50-200 μl. If the elution effect is unsatisfactory, you can add RNase-free ddH2O preheated at 65 ℃, then prolong the time of room temperature, and centrifuge the column for the second elution.
2. gDNA-Filter Columns clogging problem.
(1) Too much sample. This kit is suitable for extracting 10-20 mg of animal tissues or 2-5×106 cells, excessive amount of tissues will reduce the yield as well as the purity, and the amount of samples used should be reduced during operation.
(2) The sample is rich in muscle fibers. Muscle, heart and skin are rich in myofibers, the molecular weight of myofibers is large, which will cause blockage of the column, and the use of liquid nitrogen milling will reduce the blockage phenomenon when processing samples.
(3) Inadequate tissue grinding or homogenization. When the grinding or homogenization is not sufficient, the debris may cause the column to be blocked. The lysed liquid should be centrifuged at 13,000×g for 5 min, and the supernatant should be added to the gDNA-Filter Columns.
3. RNA degradation.
RNA degradation after purification is related to the quality of the sample, whether it is contaminated by RNase, the operation mode and other factors:
(1) Samples cause RNA degradation because they are not stored in time. Tissue samples or cells are stored at -80℃ after liquid nitrogen flash-freezing if they are not collected in time for subsequent experiments.
(2) Samples were repeatedly frozen and thawed. When storing tissue samples, store them in small pieces to avoid degradation due to repeated freezing and thawing.
(3) Electrophoresis. Common RNA degradation is partly caused by the electrophoresis process. Before electrophoresis, soak the electrophoresis tank in 3% hydrogen peroxide for 20 min, and then rinse it with RNase-free ddH2O, and configure the electrophoresis buffer with RNase-free ddH2O.
(4) Ensure that the lance and centrifuge tubes used in the extraction process are RNase-free.
4.The extracted RNA was contaminated with genomic DNA.
The DNA and RNA content of different kinds of tissues and cells varies greatly, please do not exceed 20 mg of tissue and 5×106 cells. If liver, spleen and kidney are rich in DNA, please do not exceed 10 mg, otherwise it will lead to excessive genomic residues. gDNA-Filter Columns can effectively remove most of the DNA in the system, if the downstream experiments are very sensitive to trace amounts of DNA, you can choose to select the use of the following options according to the specific circumstances:
(1) Use DNase I provided in the kit for on-membrane digestion to remove DNA contamination.
(2) Use trans-intron primers when designing primers, thus avoiding the involvement of genomic DNA templates in the amplification reaction.
(3) For reverse transcription, choose reverse transcription reagents containing a genome removal module.