Experimental Techniques

U6 is a snRNA, with a length of approximately 100 nt. Its expression abundance is usually much higher than that of most miRNAs, so the significant difference in Ct values is a normal phenomenon. However, it should be noted that:

U6 is not a true miRNA. Its length (about 100 nt) is exactly at the edge of the retention range of the conventional RNA extraction column. If the short-chain RNA enrichment step is used, U6 may be partially lost during the extraction process, resulting in the loss of reference significance for the Ct values.

In very unconventional samples such as serum, plasma, and exosomes, the content of U6 is unstable and varies greatly among individuals. It is recommended to select a more suitable reference based on the sample type (such as miR-16, miR-103, etc.).

The design should be based on the mature sequence of the target miRNA. It is recommended that the Tm value be around 55–60℃. The length of the amplification product is usually about 80 bp. If the stem-loop method is used, the reverse transcription primer can be designed by adding 6 bases to the miRNA sequence on the stem-loop sequence. Alternatively, primer design software can be used for assistance.

The amount of RNA template required for cDNA synthesis depends on individual needs. Generally, 1 μg of total RNA can be used for a routine 20-μL reverse transcription reaction (with no more than 2 μg of RNA in the 10 μL reaction system).

Gel staining method (pre-prepared gel staining): During gel preparation, directly add GelRed to the melted agarose (for example, 5 μL of 10000× stock solution for every 50 mL of gel), mix well, and then prepare the gel. Observe directly after electrophoresis. This method is simple to operate, uses less dye, and is more economical. However, GelRed has a certain impact on DNA migration, and abnormal band migration may occur when a large amount of DNA is loaded.

Bubble staining method (post-electrophoresis staining): After electrophoresis, immerse the gel in the diluted nucleic acid dye (for example, prepare a 3× working solution by diluting 10000× stock solution with 0.1M NaCl water solution at a ratio of 3300:1), and let it stain at room temperature for about 30 minutes. No decolorization is required for observation. This method has higher sensitivity and can eliminate the interference of the dye on DNA migration, but it requires more dye, and the staining solution can be reused 2-3 times.

It is recommended to use the gel staining method as the routine for experiments; for experiments with higher band separation requirements or when the loading volume is large, the bubble staining method is recommended.

Possible causes and solutions are as follows:

Excessive DNA loading: The recommended loading amount is 50–200 ng per lane. For samples of unknown concentration, try reducing the loading to 1/2 or 1/3 of the usual amount.

Too high concentration of GelRed: Try using 0.5× working concentration instead of 1×.

Inappropriate gel concentration: For high-molecular-weight DNA, a lower percentage of agarose gel is recommended.

Incompatible electrophoresis buffer: TBE buffer usually provides better separation than TAE buffer.

Switch to staining method: If the above adjustments are ineffective, it is recommended to switch to the staining method to avoid the interference of the dye on DNA migration.

In each reaction system (10 μL), the molar ratio of the vector to each insert fragment should be approximately between 1:2.5 and 1:3, and the total amount should be ≤ 200 ng.

Whether it is single-fragment or multi-fragment seamless cloning, if the length of a single fragment is ≥ 4000 bp, the total amount of nucleic acids in the reaction system should be reduced to below 100 ng. 

For multi-fragment seamless cloning, the homologous arms between the fragments can also be appropriately extended (e.g., 35 bp - 50 bp) to improve the correct recombination rate. Additionally, it is necessary to ensure that the vector used is completely linearized.

The number of transformation units is inversely proportional to the number of inserted fragments and the total length. It is advisable to use Zymagen5α competent cells for the transformation experiment, which can significantly increase the number of transformation units.

The mutation site depends on the primer design. However, for base changes of more than 50 bp, if the restriction enzyme cutting site is suitable, it is recommended to attempt seamless cloning.